Heterocyclic glutaminase inhibitors

ABSTRACT

The invention relates to novel heterocyclic compounds and pharmaceutical preparations thereof. The invention further relates to methods of treatment using the novel heterocyclic compounds of the invention.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/727,195, filed Nov. 16, 2012, and U.S.Provisional Patent Application No. 61/824,434, filed May 17, 2013, whichapplications are hereby incorporated by reference in their entirety.

BACKGROUND

Glutamine supports cell survival, growth and proliferation throughmetabolic and non-metabolic mechanisms. In actively proliferating cells,the metabolism of glutamine to lactate, also referred to as“glutaminolysis” is a major source of energy in the form of NADPH. Thefirst step in glutaminolysis is the deamination of glutamine to formglutamate and ammonia, which is catalyzed by the glutaminase enzyme.Thus, deamination via glutaminase is a control point for glutaminemetabolism.

Ever since Warburg's observation that ascites tumor cells exhibited highrates of glucose consumption and lactate secretion in the presence ofoxygen (Warburg, 1956), researchers have been exploring how cancer cellsutilize metabolic pathways to be able to continue activelyproliferating. Several reports have demonstrated how glutaminemetabolism supports macromolecular synthesis necessary for cells toreplicate (Curthoys, 1995; DeBardinis, 2008).

Thus, glutaminase has been theorized to be a potential therapeutictarget for the treatment of diseases characterized by activelyproliferating cells, such as cancer. The lack of suitable glutaminaseinhibitors has made validation of this target impossible. Therefore, thecreation of glutaminase inhibitors that are specific and capable ofbeing formulated for in vivo use could lead to a new class oftherapeutics.

SUMMARY OF INVENTION

The present invention provides a compound of formula I,

-   or a pharmaceutically acceptable salt thereof, wherein:-   L represents CH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂,    CH═CH, or

-    preferably CH₂CH₂, wherein any hydrogen atom of a CH or CH₂ unit    may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may    be replaced by alkyl, and any hydrogen atom of a CH₂ unit of CH₂CH₂,    CH₂CH₂CH₂ or CH₂ may be replaced by hydroxy;-   X represents S, O or CH═CH, preferably S or CH═CH, wherein any    hydrogen atom of a CH unit may be replaced by alkyl;-   Y, independently for each occurrence, represents H or CH₂O(CO)R₇;-   R₇, independently for each occurrence, represents H or substituted    or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,    heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;-   Z represents H or R₃(CO);-   R₁ and R₂ each independently represent H, alkyl, alkoxy or hydroxy;-   R₃ represents substituted or unsubstituted alkyl, hydroxyalkyl,    aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,    arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,    heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,    heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀), N(R₄)(R₅) or    OR₆, wherein any free hydroxyl group may be acylated to form C(O)R₇;-   R₄ and R₅ each independently for each occurrence represent H or    substituted or unsubstituted alkyl, hydroxyalkyl, acyl, aminoalkyl,    acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy,    aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇;-   R₆ represents substituted or unsubstituted alkyl, hydroxyalkyl,    aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl,    aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇;-   R₈, R₉ and R₁₀ each independently for each occurrence represent H or    substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino,    acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl,    alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl,    aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, or R₈ and R₉ together with the carbon to which    they are attached, form a carbocyclic or heterocyclic ring system,    wherein any free hydroxyl group may be acylated to form C(O)R₇, and    wherein at least two of R₈, R₉ and R₁₀ are not H;-   R₁₁ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,    heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the    aryl or heteroaryl ring is substituted with either —OCHF₂ or —OCF₃    and is optionally further substituted, or R₁₁ represents    C(R₁₂)(R₁₃)(R₁₄), N(R₄)(R₁₄) or OR₁₄, wherein any free hydroxyl    group may be acylated to form C(O)R₇;-   R₁₂ and R₁₃ each independently respresent H or substituted or    unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,    aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,    alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy,    aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇, and wherein both of R₁₂ and R₁₃ are not H; and-   R₁₄ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,    heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the    aryl or heteroaryl ring is substituted with either —OCHF₂ or —OCF₃    and is optionally further substituted.

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising an effectiveamount of any of the compounds described herein (e.g., a compound of theinvention, such as a compound of formula I), and one or morepharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical preparations may be for use in treating or preventing acondition or disease as described herein. In certain embodiments, thepharmaceutical preparations have a low enough pyrogen activity to besuitable for intravenous use in a human patient.

The present invention further provides methods of treating or preventingcancer, immunological or neurological diseases as described herein,comprising administering a compound of the invention.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plasma concentration of compounds 585 and 295 over timefollowing oral dosing of 50 mg/kg to female CD-1 mice.

FIG. 2 shows the plasma concentration of compounds 447 and 318 over timefollowing oral dosing of 50 mg/kg to female CD-1 mice.

FIG. 3 shows the plasma concentration of compound 670 over timefollowing oral dosing of 500, 250, 80 and 25 mg/kg, to female SpragueDawley rats.

FIG. 4 shows that oral administration of compound 670 to mice results inreduced tumor size in a H2122 lung adenocarcinoma xenograft model.

FIG. 5 shows a combination study with compound 670 and paclitaxel in aJIMT-1 triple negative breast cancer xenograft model.

FIG. 6 shows that oral administration of compound 670 to mice results inreduced tumor size in a RPMI-8226 multiple myeloma xenograft model.

FIG. 7 shows that compound 670 synergizes with pomalidomide ordexamethasone to produce an anti-tumor effect in multiple myeloma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of formula I,

-   or a pharmaceutically acceptable salt thereof, wherein:-   L represents CH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂,    CH═CH, or

-    preferably CH₂CH₂, wherein any hydrogen atom of a CH or CH₂ unit    may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may    be replaced by alkyl, and any hydrogen atom of a CH₂ unit of CH₂CH₂,    CH₂CH₂CH₂ or CH₂ may be replaced by hydroxy;-   X represents S, O or CH═CH, preferably S or CH═CH, wherein any    hydrogen atom of a CH unit may be replaced by alkyl;-   Y, independently for each occurrence, represents H or CH₂O(CO)R₇;-   R₇, independently for each occurrence, represents H or substituted    or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,    heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;-   Z represents H or R₃(CO);-   R₁ and R₂ each independently represent H, alkyl, alkoxy or hydroxy;-   R₃ represents substituted or unsubstituted alkyl, hydroxyalkyl,    aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,    arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,    heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,    heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀), N(R₄)(R₅) or    OR₆, wherein any free hydroxyl group may be acylated to form C(O)R₇;-   R₄ and R₅ each independently for each occurrence represent H or    substituted or unsubstituted alkyl, hydroxyalkyl, acyl, aminoalkyl,    acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy,    aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇;-   R₆ represents substituted or unsubstituted alkyl, hydroxyalkyl,    aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl,    aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇; and-   R₈, R₉ and R₁₀ each independently for each occurrence represent H or    substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino,    acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl,    alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl,    aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, or R₈ and R₉ together with the carbon to which    they are attached, form a carbocyclic or heterocyclic ring system,    wherein any free hydroxyl group may be acylated to form C(O)R₇, and    wherein at least two of R₈, R₉ and R₁₀ are not H;-   R₁₁ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,    heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the    aryl or heteroaryl ring is substituted with either —OCHF₂ or —OCF₃    and is optionally further substituted, or R₁₁ represents    C(R₁₂)(R₁₃)(R₁₄), N(R₄)(R₁₄) or OR₁₄, wherein any free hydroxyl    group may be acylated to form C(O)R₇;-   R₁₂ and R₁₃ each independently represent H or substituted or    unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,    aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,    alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy,    aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,    heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or    heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated    to form C(O)R₇, and wherein both of R₁₂ and R₁₃ are not H; and-   R₁₄ represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,    heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the    aryl or heteroaryl ring is substituted with either —OCHF₂ or —OCF₃    and is optionally further substituted.

In certain embodiments, the compound is not one of the following:

In certain embodiments wherein alkyl, hydroxyalkyl, amino, acylamino,aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, or heteroaryloxyalkyl are substituted, they aresubstituted with one or more substituents selected from substituted orunsubstituted alkyl, such as perfluoroalkyl (e.g., trifluoromethyl),alkenyl, alkoxy, alkoxyalkyl, aryl, aralkyl, arylalkoxy, aryloxy,aryloxyalkyl, hydroxyl, halo, alkoxy, such as perfluoroalkoxy (e.g.,trifluoromethoxy), alkoxyalkoxy, hydroxyalkyl, hydroxyalkylamino,hydroxyalkoxy, amino, aminoalkyl, alkylamino, aminoalkylalkoxy,aminoalkoxy, acylamino, acylaminoalkyl, such as perfluoro acylaminoalkyl(e.g., trifluoromethylacylaminoalkyl), acyloxy, cycloalkyl,cycloalkylalkyl, cycloalkylalkoxy, heterocyclyl, heterocyclylalkyl,heterocyclyloxy, heterocyclylalkoxy, heteroaryl, heteroarylalkyl,heteroarylalkoxy, heteroaryloxy, heteroaryloxyalkyl,heterocyclylaminoalkyl, heterocyclylaminoalkoxy, amido, amidoalkyl,amidine, imine, oxo, carbonyl (such as carboxyl, alkoxycarbonyl, formyl,or acyl, including perfluoroacyl (e.g., C(O)CF₃)), carbonylalkyl (suchas carboxyalkyl, alkoxycarbonylalkyl, formylalkyl, or acylalkyl,including perfluoroacylalkyl (e.g., -alkylC(O)CF₃)), carbamate,carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate, sulfamoyl, sulfone,sulfonamide, sulfonamidealkyl, cyano, nitro, azido, sulfhydryl,alkylthio, thiocarbonyl (such as thioester, thioacetate, orthioformate), phosphoryl, phosphate, phosphonate or phosphinate.

In certain embodiments, R₁₁ represents arylalkyl, such as benzyl,wherein the aryl group is substituted with —OCF₃, such asmeta-substituted with —OCF₃. In certain such embodiments, the aryl ringis not further substituted. In certain embodiments, R₁₁ representstrifluoromethoxybenzyl, such as

In certain embodiments, L represents CH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂,CH₂S, SCH₂, or CH₂NHCH₂, wherein any hydrogen atom of a CH₂ unit may bereplaced by alkyl or alkoxy, and any hydrogen atom of a CH₂ unit ofCH₂CH₂, CH₂CH₂CH₂ or CH₂ may be replaced by hydroxyl. In certainembodiments, L represents CH₂SCH₂, CH₂CH₂, CH₂S or SCH₂. In certainembodiments, L represents CH₂CH₂. In certain embodiments, L is notCH₂SCH₂.

In certain embodiments, Y represents H.

In certain embodiments, X represents S or CH═CH. In certain embodiments,X represents S.

In certain embodiments, Z represents R₃(CO). In certain embodimentswherein Z is R₃(CO), R₃ and R₁₁ are not identical (e.g., the compound offormula I is not symmetrical).

In certain embodiments, R₁ and R₂ each represent H.

In certain embodiments, Z represents R₃(CO) and R₃ represents arylalkyl,heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain embodiments,Z represents R₃(CO) and R₃ represents heteroarylalkyl, such aspyridylalkyl (e.g., pyridylmethyl). In certain such embodiments, Zrepresents

In certain embodiments, Z represents R₃(CO) and R₃ representsC(R₈)(R₉)(R₁₀), wherein R₈ represents aryl, arylalkyl, heteroaryl orheteroaralkyl, such as aryl, arylalkyl or heteroaryl, R₉ represents H,and R₁₀ represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such ashydroxy, hydroxyalkyl or alkoxy.

In certain embodiments, L represents CH₂SCH₂, CH₂CH₂, CH₂S or SCH₂, suchas CH₂CH₂, Y represents H, X represents S, Z represents R₃(CO), R₁ andR₂ each represent H, R₃ represents arylalkyl, heteroarylalkyl,cycloalkyl or heterocycloalkyl, such as heteroarylalkyl (e.g.,pyridylalkyl), and R₁₁ represents arylalkyl, such trifluoromethoxybenzyl(e.g.,

In certain such embodiments, Z represents R₃(CO) and R₃ representspyridylmethyl, such as wherein Z represents

In certain embodiments, L represents CH₂SCH₂, CH₂CH₂, CH₂S or SCH₂, suchas CH₂CH₂, Y represents H, X represents S, Z represents R₃(CO), R₁ andR₂ each represent H, and each R₃ represents C(R₈)(R₉)(R₁₀), wherein R₈represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl,arylalkyl or heteroaryl, R₉ represents H, and R₁₀ represents hydroxy,hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl oralkoxy, and R₁₁ represents arylalkyl, such trifluoromethoxybenzyl (e.g.,

In certain embodiments, L represents CH₂CH₂, Y represents H, Xrepresents S or CH═CH, such as S, Z represents R₃(CO), R₁ and R₂ eachrepresent H, R₃ represents substituted or unsubstituted arylalkyl,heteroarylalkyl, cycloalkyl or heterocycloalkyl, such as heteroarylalkyl(e.g., pyridylalkyl), and R₁₁ represents arylalkyl, suchtrifluoromethoxybenzyl (e.g.,

In certain such embodiments, Z represents R₃(CO) and R₃ representspyridylmethyl, such as wherein Z represents

In certain embodiments, L represents CH₂CH₂, Y represents H, Xrepresents S, Z represents R₃(CO), R₁ and R₂ each represent H, h R₃represents C(R₈)(R₉)(R₁₀), wherein R₈ represents aryl, arylalkyl orheteroaryl, R₉ represents H, and R₁₀ represents hydroxy, hydroxyalkyl oralkoxy, and R₁₁ represents arylalkyl, such trifluoromethoxybenzyl (e.g.,

In certain such embodiments, R₈ represents aryl and R₁₀ representshydroxyalkyl.

In certain embodiments, the compound is selected from compound 447, 585,586, 600, 614, 615, 629, 636, 657, 658, 659, 660, 661, 662, 663, 666,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,682, 683, 684, 685, 686, 687, 688, 689, 690, 692, 693, 694, 695, 696,697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 715,716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, or730. In certain embodiments, the compound is selected from compound 657,658, 659, 660, 661, 662, 663, 666, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,689, 690, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703,704, 705, 706, 707, 708, 709, 715, 716, 717, 718, 719, 720, 721, 722,723, 724, 725, 726, 727, 728, 729, or 730.

In certain embodiments, compounds of the invention may be prodrugs ofthe compounds of formula I, e.g., wherein a hydroxyl in the parentcompound is presented as an ester or a carbonate, or carboxylic acidpresent in the parent compound is presented as an ester. In certain suchembodiments, the prodrug is metabolized to the active parent compound invivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, orcarboxylic acid).

In certain embodiments, compounds of the invention may be racemic. Incertain embodiments, compounds of the invention may be enriched in oneenantiomer. For example, a compound of the invention may have greaterthan 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95%or greater ee. In certain embodiments, compounds of the invention mayhave more than one stereocenter. In certain such embodiments, compoundsof the invention may be enriched in one or more diastereomer. Forexample, a compound of the invention may have greater than 30% de, 40%de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.

In certain embodiments, the present invention relates to methods oftreatment with a compound of formula I, or a pharmaceutically acceptablesalt thereof. In certain embodiments, the therapeutic preparation may beenriched to provide predominantly one enantiomer of a compound (e.g., offormula I). An enantiomerically enriched mixture may comprise, forexample, at least 60 mol percent of one enantiomer, or more preferablyat least 75, 90, 95, or even 99 mol percent. In certain embodiments, thecompound enriched in one enantiomer is substantially free of the otherenantiomer, wherein substantially free means that the substance inquestion makes up less than 10%, or less than 5%, or less than 4%, orless than 3%, or less than 2%, or less than 1% as compared to the amountof the other enantiomer, e.g., in the composition or compound mixture.For example, if a composition or compound mixture contains 98 grams of afirst enantiomer and 2 grams of a second enantiomer, it would be said tocontain 98 mol percent of the first enantiomer and only 2% of the secondenantiomer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one diastereomer of a compound (e.g., of formulaI). A diastereomerically enriched mixture may comprise, for example, atleast 60 mol percent of one diastereomer, or more preferably at least75, 90, 95, or even 99 mol percent.

In certain embodiments, the present invention relates to methods oftreatment with a compound of formula I, or a pharmaceutically acceptablesalt thereof. In certain embodiments, the therapeutic preparation may beenriched to provide predominantly one enantiomer of a compound (e.g., offormula I). An enantiomerically enriched mixture may comprise, forexample, at least 60 mol percent of one enantiomer, or more preferablyat least 75, 90, 95, or even 99 mol percent. In certain embodiments, thecompound enriched in one enantiomer is substantially free of the otherenantiomer, wherein substantially free means that the substance inquestion makes up less than 10%, or less than 5%, or less than 4%, orless than 3%, or less than 2%, or less than 1% as compared to the amountof the other enantiomer, e.g., in the composition or compound mixture.For example, if a composition or compound mixture contains 98 grams of afirst enantiomer and 2 grams of a second enantiomer, it would be said tocontain 98 mol percent of the first enantiomer and only 2% of the secondenantiomer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one diastereomer of a compound (e.g., of formulaI). A diastereomerically enriched mixture may comprise, for example, atleast 60 mol percent of one diastereomer, or more preferably at least75, 90, 95, or even 99 mol percent.

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising any of thecompounds shown above (e.g., a compound of the invention, such as acompound of formula I), and one or more pharmaceutically acceptableexcipients. In certain embodiments, the pharmaceutical preparations maybe for use in treating or preventing a condition or disease as describedherein. In certain embodiments, the pharmaceutical preparations have alow enough pyrogen activity to be suitable for use in a human patient.

Compounds of any of the above structures may be used in the manufactureof medicaments for the treatment of any diseases or conditions disclosedherein.

Uses of Enzyme Inhibitors Glutamine plays an important role as a carrierof nitrogen, carbon, and energy. It is used for hepatic urea synthesis,for renal ammoniagenesis, for gluconeogenesis, and as respiratory fuelfor many cells. The conversion of glutamine into glutamate is initatedby the mitochondrial enzyme, glutaminase (“GLS”). There are two majorforms of the enzyme, K-type and L-type, which are distinguished by theirKm values for glutamine and response to glutamate, wherein the Km value,or Michaelis constant, is the concentration of substrate required toreach half the maximal velocity. The L-type, also known as “liver-type”or GLS2, has a high Km for glutamine and is glutamate resistant. TheK-type, also known as “kidney-type or GLS1, has a low Km for glutamineand is inhibited by glutamate. An alternative splice form of GLS1,referred to as glutmainase C or “GAC”, has been identified recently andhas similar activity characteristics of GLS1. In certain embodiments,the compounds may selectively inhibit GLS1, GLS2 and GAC. In a preferredembodiment, the compounds selectively inhibit GLS1 and GAC.

In addition to serving as the basic building blocks of proteinsynthesis, amino acids have been shown to contribute to many processescritical for growing and dividing cells, and this is particularly truefor cancer cells. Nearly all definitions of cancer include reference todysregulated proliferation. Numerous studies on glutamine metabolism incancer indicate that many tumors are avid glutamine consumers (Souba,Ann. Surg., 1993; Collins et al., J. Cell. Physiol., 1998; Medina, J.Nutr., 2001; Shanware et al., J. Mol. Med., 2011). An embodiment of theinvention is the use of the compounds described herein for the treatmentof cancer.

In certain embodiments, the cancer may be one or a variant of AcuteLymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma andLymphoma), Anal Cancer, Appendix Cancer, Atypical Teratoid/RhabdoidTumor, Basal Cell Carcinoma, Bile Duct Cancer (including Extrahepatic),Bladder Cancer, Bone Cancer (including Osteosarcoma and MalignantFibrous Histiocytoma), Brain Tumor (such as Astrocytomas, Brain andSpinal Cord Tumors, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors,Craniopharyngioma, Ependymoblastoma, Ependymoma, Medulloblastoma,Medulloepithelioma, Pineal Parenchymal Tumors of IntermediateDifferentiation, Supratentorial Primitive Neuroectodermal Tumors andPineoblastoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, BasalCell Carcinoma, Bile Duct Cancer (including Extrahepatic), BladderCancer, Bone Cancer (including Osteosarcoma and Malignant FibrousHistiocytoma), Carcinoid Tumor, Carcinoma of Unknown Primary, CentralNervous System (such as Atypical Teratoid/Rhabdoid Tumor, EmbryonalTumors and Lymphoma), Cervical Cancer, Childhood Cancers, Chordoma,Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma (Mycosis Fungoides andSézary Syndrome), Duct, Bile (Extrahepatic), Ductal Carcinoma In Situ(DCIS), Embryonal Tumors (Central Nervous System), Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer(like Intraocular Melanoma, Retinoblastoma), Fibrous Histiocytoma ofBone (including Malignant and Osteosarcoma) Gallbladder Cancer, Gastric(Stomach) Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalStromal Tumors (GIST), Germ Cell Tumor (Extracranial, Extragonadal,Ovarian), Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia,Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors (Endocrine, Pancreas), KaposiSarcoma, Kidney (including Renal Cell), Langerhans Cell Histiocytosis,Laryngeal Cancer, Leukemia (including Acute Lymphoblastic (ALL), AcuteMyeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML),Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), LobularCarcinoma In Situ (LCIS), Lung Cancer (Non-Small Cell and Small Cell),Lymphoma (AIDS-Related, Burkitt, Cutaneous T-Cell (Mycosis Fungoides andSézary Syndrome), Hodgkin, Non-Hodgkin, Primary Central Nervous System(CNS), Macroglobulinemia, Waldenström, Male Breast Cancer, MalignantFibrous Histiocytoma of Bone and Osteosarcoma, Medulloblastoma,Medulloepithelioma, Melanoma (including Intraocular (Eye)), Merkel CellCarcinoma, Mesothelioma (Malignant), Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia,Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma and MultipleMyeloma, Myeloproliferative Disorders (Chronic), Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin Lymphoma (both B-cell and T-cell subtypes), Non-Small CellLung Cancer, Oral Cancer, Oral Cavity Cancer, Lip and, OropharyngealCancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, OvarianCancer (such as Epithelial, Germ Cell Tumor, and Low Malignant PotentialTumor), Pancreatic Cancer (including Islet Cell Tumors), Papillomatosis,Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, ParathyroidCancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, PinealParenchymal Tumors of Intermediate Differentiation, Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, PlasmaCell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy andBreast Cancer, Primary Central Nervous System (CNS) Lymphoma, ProstateCancer, Rectal Cancer, Renal Cell (Kidney) Cancer (RCC), Renal Pelvisand Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma,Salivary Gland Cancer, Sarcoma (like Ewing Sarcoma Family of Tumors,Kaposi, Soft Tissue, Uterine), Sézary Syndrome, Skin Cancer (such asMelanoma, Merkel Cell Carcinoma, Nonmelanoma), Small Cell Lung Cancer,Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma,Squamous cell carcinoma of the head and neck (HNSCC), Squamous NeckCancer with Occult Primary, Metastatic, Stomach (Gastric) Cancer,Supratentorial Primitive Neuroectodermal Tumors, T-Cell Lymphoma(Cutaneous, Mycosis Fungoides and Sézary Syndrome), Testicular Cancer,Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer,Transitional Cell Cancer of the Renal Pelvis and Ureter, Triple NegativeBreast Cancer (TNBC), Trophoblastic Tumor (Gestational), UnknownPrimary, Unusual Cancers of Childhood, Ureter and Renal Pelvis,Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial,Uterine Sarcoma, Waldenström Macroglobulinemia and Wilms Tumor. FIGS. 4,5 and 6 show that a compound of the invention reduces tumor sizes inxenograft models of lung adenocarcinoma, breast cancer and multiplemyeloma, demonstrating that the compounds described herein may be usedfor the treatment of a variety of cancers.

In some instances, oncogenic mutations promote glutamine metabolism.Cells expressing oncogenic K-Ras exhibt increased ultilization ofglutamine (Weinberg et al., Proc. Natl. Acad. Sci. USA, 2010; Gaglio etal., Mol. Syst. Biol., 2011). In certain embodiments, the cancer cellshave a mutated K-Ras gene. In certain embodiments, the cancer isassociated with tissue of the bladder, bone marrow, breast, colon,liver, lung, ovary, pancreas, prostate, skin or thyroid. The c-Myc geneis known to be altered in numerous cancers (Zeller et al., Genomebiology, 2003). Increased Myc protein expression has been correlatedwith increased expression of glutaminase, leading to up-regulation ofglutamine metabolism (Dang et al., Clin. Cancer Res., 2009; Gao et al.,Nature, 2009). In certain embodiments, the cancer cells have anoncogenic c-Myc gene or elevated Myc protein expression. In someembodiments, the cancer is associated with tissue of the bladder, bone,bowel, breast, central nervous system (like brain), colon, gastricsystem (such as stomach and intestine), liver, lung, ovary, prostate,muscle, and skin.

While many cancer cells depend on exogenous glutamine for survival, thedegree of glutamine dependence among tumor cell subtypes may make apopulation of cells more susceptible to the reduction of glutamine. Asan example, gene expression analysis of breast cancers has identifiedfive intrinsic subtypes (luminal A, luminal B, basal, HER2+, andnormal-like) (Sorlie et al., Proc Natl Acad Sci USA, 2001). Althoughglutamine deprivation has an impact on cell growth and viability,basal-like cells appear to be more sensitive to the reduction ofexogenous glutamine (Kung et al., PLoS Genetics, 2011). This supportsthe concept that glutamine is a very important energy source inbasal-like breast cancer cell lines, and suggests that inhibition of theglutaminase enzyme would be beneficial in the treatment of breastcancers comprised of basal-like cells. Triple-negative breast cancer(TNBC) is characterized by a lack of estrogen receptor, progesteronereceptor and human epidermal growth factor receptor 2 expression. It hasa higher rate of relapse following chemotherapy, and a poorer prognosisthan with the other breast cancer subtypes (Dent et al., Clin Cancerres, 2007). Interestingly, there appears to be significant similaritiesin metabolic profiling between TNBC cells and basal-like breast cancercells (unpublished data). FIG. 5 shows that a compound as describedherein reduces a TNBC xenograft tumor. The compound in combination withpaclitaxel further reduced the tumor size. Therefore, the inventioncontemplates the use of the compounds described herein for the treatmentof TNBC and basal-type breast cancers.

Cachexia, the massive loss of muscle mass, is often associated with poorperformance status and high mortality rate of cancer patients. A theorybehind this process is that tumors require more glutamine than isnormally supplied by diet, so muscle, a major source of glutamine,starts to breakdown in order to supply enough nutrient to the tumor.Thus, inhibition of glutaminase may reduce the need to breakdown muscle.An embodiment of the invention is the use of the present compounds toprevent, inhibit or reduce cachexia.

The most common neurotransmitter is glutamate, derived from theenzymatic conversion of glutamine via glutaminase. High levels ofglutamate have been shown to be neurotoxic. Following traumatic insultto neuronal cells, there occurs a rise in neurotransmitter release,particularly glutamate. Accordingly, inhibition of glutaminase has beenhypothesized as a means of treatment following an ischemic insult, suchas stroke (Newcomb, PCT WO 99/09825, Kostandy, Neurol. Sci., 2011).Huntington's disease is a progressive, fatal neurological condition. Ingenetic mouse models of Huntington's disease, it was observed that theearly manifestation of the disease correlated with dysregulatedglutamate release (Raymond et al., Neuroscience, 2011). InHIV-associated dementia, HIV infected macrophages exhibit upregulatedglutaminase activity and increased glutamate release, leading toneuronal damage (Huang et al., J Neurosci., 2011). Similarly, in anotherneurological disease, the activated microglia in Rett Syndrome releaseglutamate causing neuronal damage. The release of excess glutamate hasbeen associated with the up-regulation of glutaminase (Maezawa et al.,J. Neurosci, 2010). In mice bred to have reduced glutaminase levels,sensitivity to psychotic-stimulating drugs, such as amphetamines, wasdramatically reduced, thus suggesting that glutaminase inhibition may bebeneficial in the treatment of schizophrenia (Gaisler-Salomon et al.,Neuropsychopharmacology, 2009). Bipolar disorder is a devastatingillness that is marked by recurrent episodes of mania and depression.This disease is treated with mood stabilizers such as lithium andvalproate; however, chronic use of these drugs appear to increase theabundance of glutamate receptors (Nanavati et al., J. Neurochem., 2011),which may lead to a decrease in the drug's effectiveness over time.Thus, an alternative treatment may be to reduce the amount of glutamateby inhibiting glutaminase. This may or may not be in conjunction withthe mood stabilizers. Memantine, a partial antagonist ofN-methyl-D-aspartate receptor (NMDAR), is an approved therapeutic in thetreatment of Alzheimer's disease. Currently, research is being conductedlooking at memantine as a means of treating vascular dementia andParkinson's disease (Oliverares et al., Curr. Alzheimer Res., 2011).Since memantine has been shown to partially block the NMDA glutamatereceptor also, it is not unresasonable to speculate that decreasingglutamate levels by inhibiting glutaminase could also treat Alzheimer'sdisease, vascular dementia and Parkinson's disease. Alzheimer's disease,bipolar disorder, HIV-associated dementia, Huntington's disease,ischemic insult, Parkinson's disease, schizophrenia, stroke, traumaticinsult and vascular dementia are but a few of the neurological diseasesthat have been correlated to increased levels of glutamate. Thus,inhibiting glutaminase with a compound described herein can reduce orprevent neurological diseases. Therefore, in one embodiment, thecompounds may be used for the treatment or prevention of neurologicaldiseases.

Activation of T lymphocytes induces cell growth, proliferation, andcytokine production, thereby placing energetic and biosynthetic demandson the cell. Glutamine serves as an amine group donor for nucleotidesynthesis, and glutamate, the first component in glutamine metabolism,plays a direct role in amino acid and glutathione synthesis, as well asbeing able to enter the Krebs cycle for energy production (Carr et al.,J. Immunol., 2010). Mitogen-induced T cell proliferation and cytokineproduction require high levels of glutamine metabolism, thus inhibitingglutaminase may serve as a means of immune modulation. In multiplesclerosis, an inflammatory autoimmune disease, the activated microgliaexhibit up-regulated glutaminase and release increased levels ofextracellular glutamate. Glutamine levels are lowered by sepsis, injury,burns, surgery and endurance exercise (Calder et al., Amino Acids,1999). These situations put the individual at risk of immunosuppression.In fact, in general, glutaminase gene expression and enzyme activity areboth increased during T cell activity. Patients given glutaminefollowing bone marrow transplantation resulted in a lower level ofinfection and reduced graft v. host disease (Crowther, Proc. Nutr. Soc.,2009). T cell proliferation and activiation is involved in manyimmunological diseases, such as inflammatory bowel disease, Crohn'sdisease, sepsis, psoriasis, arthritis (including rheumatoid arthritis),multiple sclerosis, graft v. host disease, infections, lupus anddiabetes. In an embodiment of the invention, the compounds describedherein can be used to treat or prevent immunological diseases.

Hepatic encephalopathy (HE) represents a series of transient andreversible neurologic and psychiatric dysfunction in patients with liverdisease or portosystemic shunting. HE is not a single clinical entityand may reflect reversible metabolic encephalopathy, brain atrophy,brain edema, or a combination of these factors; however, the currenthypothesis is that the accumulation of ammonia, mostly derived from theintestine, plays a key role in the pathophysiology (Khunger et al., ClinLiver Dis, 2012). The deamination of glutamine in small intestine, renaland muscle synthesis all contribute to ammonia production. Impairedhepatic clearance caused by hepatocellular clearance or portosystemicshunting causes increased accumulation of ammonia Ammonia toxicityaffects astrocytes in the brain via glutamine synthetase, whichmetabolizes the ammonia to produce increased glutamine. Glutamine, inturn, attracts water into the astrocytes, leading to swelling andoxidative dysfunction of the mitochondria. The resulting cerebral edemais thought to contribute to neurologic dysfunction seen in HE (Kavitt etal., Clin Gastroenterol Hepatol, 2008). In an embodiment of theinvention, the compounds described herein can be used to treat orprevent HE.

Primary sensory neurons in the dorsal root ganglion have been shown toelevate their glutaminase enzyme activity following inflammation (Milleret al., Pain Research and Treatment, 2012). It is believed that theresulting increased glutamate production contributes to both central andperipheral sensitization, identified as pain. An aspect of the inventionis the use of the present compounds herein for the treatment ordiminishment of pain. In certain embodiments, the pain can beneuropathic pain, chemotherapy-induced pain or inflammatory pain.

High blood glucose levels, high insulin levels, and insulin resistanceare risk factors for developing diabetes mellitus. Similarly, high bloodpressure is a risk factor for developing cardiovascular disease. In arecent report from a large human cohort study, these four risk factorswere inversely correlated with glutamine-to-glutamate ratios in theblood stream (Chen et al, Circulation, 2012). Furthermore, plasmaglutamine-to-glutamate ratios were inversely correlated with theeventual incidence of diabetes mellitus over 12 years (Cheng et al,Circulation, 2012). Experiments with animal models were consistent withthese findings. Mice fed glutamine-rich diets exhibited lower bloodglucose levels in a glucose tolerance test after 6 hours of fasting, andintraperitoneal injection of glutamine into mice rapidly decreased theirblood pressure (Cheng et al, Circulation, 2012). Therefore, it isplausible that glutaminase inhibitors, which cause increased glutaminelevels and decrease glutamate levels, would decrease the incidence ofdiabetes mellitus and cardiovascular disease. In particular, the liverand small intestine are major sites of glutamine utilization in diabeticanimals, and glutaminase activity is higher than normal in these organsin streptozotocin-induced diabetic rats (Watford et al, Biochem J, 1984;Mithieux et al, Am J Physiol Endrocrinol Metab, 2004). In an embodimentof the invention, the compounds described herein can be used to treatdiabetes. In another embodiment of the invention, the present compoundscan be used to reduce high blood pressure.

In one embodiment, the method of treating or preventing cancer,immunological and neurological diseases may comprise administering acompound of the invention conjointly with a chemotherapeutic agent.Chemotherapeutic agents that may be conjointly administered withcompounds of the invention include: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, bortezomib,buserelin, busulfan, campothecin, capecitabine, carboplatin,carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin,cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin,dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol,docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide,everolimus, exemestane, filgrastim, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein,goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon,irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide,levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide,oxaliplatin, paclitaxel, pamidronate, pentostatin, perifosine,plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed,rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen,temozolomide, temsirolimus, teniposide, testosterone, thalidomide,thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab,tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

Many combination therapies have been developed for the treatment ofcancer. In certain embodiments, compounds of the invention may beconjointly administered with a combination therapy. Examples ofcombination therapies with which compounds of the invention may beconjointly administered are included in Table 1.

TABLE 1 Exemplary combinatorial therapies for the treatment of cancer.Name Therapeutic agents ABV Doxorubicin, Bleomycin, Vinblastine ABVDDoxorubicin, Bleomycin, Vinblastine, Dacarbazine AC (Breast)Doxorubicin, Cyclophosphamide AC (Sarcoma) Doxorubicin, Cisplatin AC(Neuroblastoma) Cyclophosphamide, Doxorubicin ACE Cyclophosphamide,Doxorubicin, Etoposide ACe Cyclophosphamide, Doxorubicin AD Doxorubicin,Dacarbazine AP Doxorubicin, Cisplatin ARAC-DNR Cytarabine, DaunorubicinB-CAVe Bleomycin, Lomustine, Doxorubicin, Vinblastine BCVPP Carmustine,Cyclophosphamide, Vinblastine, Procarbazine, Prednisone BEACOPPBleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine,Procarbazine, Prednisone, Filgrastim BEP Bleomycin, Etoposide, CisplatinBIP Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP Bleomycin, Vincristine,Cisplatin, Mitomycin CA Cytarabine, Asparaginase CABO Cisplatin,Methotrexate, Bleomycin, Vincristine CAF Cyclophosphamide, Doxorubicin,Fluorouracil CAL-G Cyclophosphamide, Daunorubicin, Vincristine,Prednisone, Asparaginase CAMP Cyclophosphamide, Doxorubicin,Methotrexate, Procarbazine CAP Cyclophosphamide, Doxorubicin, CisplatinCaT Carboplatin, Paclitaxel CAV Cyclophosphamide, Doxorubicin,Vincristine CAVE ADD CAV and Etoposide CA-VP16 Cyclophosphamide,Doxorubicin, Etoposide CC Cyclophosphamide, Carboplatin CDDP/VP-16Cisplatin, Etoposide CEF Cyclophosphamide, Epirubicin, FluorouracilCEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or without/Bleomycin CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin,Fluorouracil or Carboplatin Fluorouracil CHAP Cyclophosphamide orCyclophosphamide, Altretamine, Doxorubicin, Cisplatin ChlVPPChlorambucil, Vinblastine, Procarbazine, Prednisone CHOPCyclophosphamide, Doxorubicin, Vincristine, Prednisone CHOP-BLEO AddBleomycin to CHOP CISCA Cyclophosphamide, Doxorubicin, CisplatinCLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin CMF Methotrexate,Fluorouracil, Cyclophosphamide CMFP Cyclophosphamide, Methotrexate,Fluorouracil, Prednisone CMFVP Cyclophosphamide, Methotrexate,Fluorouracil, Vincristine, Prednisone CMV Cisplatin, Methotrexate,Vinblastine CNF Cyclophosphamide, Mitoxantrone, Fluorouracil CNOPCyclophosphamide, Mitoxantrone, Vincristine, Prednisone COB Cisplatin,Vincristine, Bleomycin CODE Cisplatin, Vincristine, Doxorubicin,Etoposide COMLA Cyclophosphamide, Vincristine, Methotrexate, Leucovorin,Cytarabine COMP Cyclophosphamide, Vincristine, Methotrexate, PrednisoneCooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil,Vincristine, Prednisone COP Cyclophosphamide, Vincristine, PrednisoneCOPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPPCyclophosphamide, Vincristine, Procarbazine, Prednisone CP(ChronicChlorambucil, Prednisone lymphocytic leukemia) CP (Ovarian Cancer)Cyclophosphamide, Cisplatin CT Cisplatin, Paclitaxel CVD Cisplatin,Vinblastine, Dacarbazine CVI Carboplatin, Etoposide, Ifosfamide, MesnaCVP Cyclophosphamide, Vincristine, Prednisome CVPP Lomustine,Procarbazine, Prednisone CYVADIC Cyclophosphamide, Vincristine,Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine DAT Daunorubicin,Cytarabine, Thioguanine DAV Daunorubicin, Cytarabine, Etoposide DCTDaunorubicin, Cytarabine, Thioguanine DHAP Cisplatin, Cytarabine,Dexamethasone DI Doxorubicin, Ifosfamide DTIC/Tamoxifen Dacarbazine,Tamoxifen DVP Daunorubicin, Vincristine, Prednisone EAP Etoposide,Doxorubicin, Cisplatin EC Etoposide, Carboplatin EFP Etoposie,Fluorouracil, Cisplatin ELF Etoposide, Leucovorin, Fluorouracil EMA 86Mitoxantrone, Etoposide, Cytarabine EP Etoposide, Cisplatin EVAEtoposide, Vinblastine FAC Fluorouracil, Doxorubicin, CyclophosphamideFAM Fluorouracil, Doxorubicin, Mitomycin FAMTX Methotrexate, Leucovorin,Doxorubicin FAP Fluorouracil, Doxorubicin, Cisplatin F-CL Fluorouracil,Leucovorin FEC Fluorouracil, Cyclophosphamide, Epirubicin FEDFluorouracil, Etoposide, Cisplatin FL Flutamide, Leuprolide FZFlutamide, Goserelin acetate implant HDMTX Methotrexate, LeucovorinHexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil ICE-TIfosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna IDMTX/6-MPMethotrexate, Mercaptopurine, Leucovorin IE Ifosfamide, Etoposie, MesnaIfoVP Ifosfamide, Etoposide, Mesna IPA Ifosfamide, Cisplatin,Doxorubicin M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone,Melphalan MAC-III Methotrexate, Leucovorin, Dactinomycin,Cyclophosphamide MACC Methotrexate, Doxorubicin, Cyclophosphamide,Lomustine MACOP-B Methotrexate, Leucovorin, Doxorubicin,Cyclophosphamide, Vincristine, Bleomycin, Prednisone MAID Mesna,Doxorubicin, Ifosfamide, Dacarbazine m-BACOD Bleomycin, Doxorubicin,Cyclophosphamide, Vincristine, Dexamethasone, Methotrexate, LeucovorinMBC Methotrexate, Bleomycin, Cisplatin MC Mitoxantrone, Cytarabine MFMethotrexate, Fluorouracil, Leucovorin MICE Ifosfamide, Carboplatin,Etoposide, Mesna MINE Mesna, Ifosfamide, Mitoxantrone, Etoposidemini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan MOBP Bleomycin,Vincristine, Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine,Procarbazine MOPP Mechlorethamine, Vincristine, Procarbazine, PrednisoneMOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,Doxorubicin, Bleomycin, Vinblastine MP (multiple Melphalan, Prednisonemyeloma) MP (prostate cancer) Mitoxantrone, Prednisone MTX/6-MOMethotrexate, Mercaptopurine MTX/6-MP/VP Methotrexate, Mercaptopurine,Vincristine, Prednisone MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin,Doxorubicin MV (breast cancer) Mitomycin, Vinblastine MV (acutemyelocytic Mitoxantrone, Etoposide leukemia) M-VAC MethotrexateVinblastine, Doxorubicin, Cisplatin MVP Mitomycin Vinblastine, CisplatinMVPP Mechlorethamine, Vinblastine, Procarbazine, Prednisone NFLMitoxantrone, Fluorouracil, Leucovorin NOVP Mitoxantrone, Vinblastine,Vincristine OPA Vincristine, Prednisone, Doxorubicin OPPA AddProcarbazine to OPA. PAC Cisplatin, Doxorubicin PAC-I Cisplatin,Doxorubicin, Cyclophosphamide PA-CI Cisplatin, Doxorubicin PCPaclitaxel, Carboplatin or Paclitaxel, Cisplatin PCV Lomustine,Procarbazine, Vincristine PE Paclitaxel, Estramustine PFL Cisplatin,Fluorouracil, Leucovorin POC Prednisone, Vincristine, Lomustine ProMACEPrednisone, Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide,Etoposide ProMACE/cytaBOM Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin,Cotrimoxazole PRoMACE/MOPP Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Mechlorethamine, Vincristine, Procarbazine, Methotrexate,Leucovorin Pt/VM Cisplatin, Teniposide PVA Prednisone, Vincristine,Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PVDA Prednisone,Vincristine, Daunorubicin, Asparaginase SMF Streptozocin, Mitomycin,Fluorouracil TAD Mechlorethamine, Doxorubicin, Vinblastine, Vincristine,Bleomycin, Etoposide, Prednisone TCF Paclitaxel, Cisplatin, FluorouracilTIP Paclitaxel, Ifosfamide, Mesna, Cisplatin TTT Methotrexate,Cytarabine, Hydrocortisone Topo/CTX Cyclophosphamide, Topotecan, MesnaVAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, BleomycinVAC Vincristine, Dactinomycin, Cyclophosphamide VACAdr Vincristine,Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine VADVincristine, Doxorubicin, Dexamethasone VATH Vinblastine, Doxorubicin,Thiotepa, Flouxymesterone VBAP Vincristine, Carmustine, Doxorubicin,Prednisone VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide,Prednisone VC Vinorelbine, Cisplatin VCAP Vincristine, Cyclophosphamide,Doxorubicin, Prednisone VD Vinorelbine, Doxorubicin VelP Vinblastine,Cisplatin, Ifosfamide, Mesna VIP Etoposide, Cisplatin, Ifosfamide, MesnaVM Mitomycin, Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide,Prednisone VP Etoposide, Cisplatin V-TAD Etoposide, Thioguanine,Daunorubicin, Cytarabine 5 + 2 Cytarabine, Daunorubicin, Mitoxantrone7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or Mitoxantrone “8 in1” Methylprednisolone, Vincristine, Lomustine, Procarbazine,Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine

The proliferation of cancer cells requires lipid synthesis. Normally,acetyl-coA used for lipid synthesis is formed from a mitochondrial poolof pyruvate that is derived from glycolysis. Yet under hypoxicconditions, such as those normally found in a tumor environment, theconversion of pyruvate to acetyl-coA within the mitochondria isdownregulated. Recent studies from Metallo et al. (2011) and Mullen etal. (2011) revealed that under such hypoxic conditions, cells insteadlargely switch to using a pathway involving the reductive carboxylationof alpha-ketoglutarate to make acetyl-coA for lipid synthesis. The firststep in this pathway involves converting glutamine to glutamate viaglutaminase enzymes. Subsequently, glutamate is converting toalpha-ketoglutarate, and the resulting alpha-ketoglutarate is convertedto isocitrate in a reductive carboxylation step mediated by theisocitrate dehydrogenase enzymes. A switch to this reductivecarboxylation pathway also occurs in some renal carcinoma cell linesthat contain either impaired mitochondria or an impaired signal forinduction of the enzyme responsible for converting glycolytic pyruvateto acetyl-coA (Mullen et al 2011). A similar switch occurs in cellsexposed to mitochondrial respiratory chain inhibitors such as metformin,rotenone, and antimycin (Mullen at al. 2011). Therefore, in someembodiments of this invention, we propose using combinations ofmitochondrial respiratory chain inhibitors and glutaminase inhibitors tosimultaneously increase cancer cells' dependence onglutaminase-dependent pathways for lipid synthesis while inhibitingthose very pathways.

The increased dependence on glycolysis in tumor cells is likely becausethe hypoxic tumor environment impairs mitochondrial respiration.Furthermore, depletion of glucose induces apoptosis in cells transformedwith the MYC oncogene. These findings suggest that inhibiting glycolysiswould have a therapeutic value in preventing cancer cell proliferation.There are currently many documented glycolytic inhibitors (Pelicano etal. 2006). However, as pointed out by Zhao et al. (2012), “availableglycolytic inhibitors are generally not very potent, and high doses arerequired, which may cause high levels of systemic toxicity.” Sincecancer cells typically use both glucose and glutamine at higher levelsthan normal cells, impairing utilization of each of those metaboliteswill likely have a synergistic effect. Therefore, in some embodiments ofthis invention, we propose using combinations of glycolytic pathwayinhibitors and glutaminase inhibitors. Such glycolytic inhibitorsinclude 2-deoxyglucose, lonidamine, 3-bromopyruvate, imatinib,oxythiamine, rapamycin, and their pharmacological equivalents.Glycolysis can be inhibited indirectly by depleting NAD+ via DNA damageinduced by DNA alkylating agents through a pathway activated bypoly(ADP-ribose) polymerase (Zong et al. 2004). Therefore, in oneembodiment of this invention, we propose using a combination of DNAalkylating agents and glutaminase inhibitors. Cancer cells use thepentose phosphate pathway along with the glycolytic pathway to createmetabolic intermediates derived from glucose. Therefore, in anotherembodiment of this invention, we propose using a combination of pentosephosphate inhibitors such as 6-aminonicotinamide along with glutaminaseinhibitors.

In certain embodiments, a compound of the invention may be conjointlyadministered with non-chemical methods of cancer treatment. In certainembodiments, a compound of the invention may be conjointly administeredwith radiation therapy. In certain embodiments, a compound of theinvention may be conjointly administered with surgery, withthermoablation, with focused ultrasound therapy, with cryotherapy, orwith any combination of these.

In certain embodiments, different compounds of the invention may beconjointly administered with one or more other compounds of theinvention. Moreover, such combinations may be conjointly administeredwith other therapeutic agents, such as other agents suitable for thetreatment of cancer, immunological or neurological diseases, such as theagents identified above. In certain embodiments, conjointlyadministering one or more additional chemotherapeutic agents with acompound of the invention provides a synergistic effect, such as shownin FIG. 7. In certain embodiments, conjointly administering one or moreadditional chemotherapeutics agents provides an additive effect.

In certain embodiments, the present invention provides a kit comprising:a) one or more single dosage forms of a compound of the invention; b)one or more single dosage forms of a chemotherapeutic agent as mentionedabove; and c) instructions for the administration of the compound of theinvention and the chemotherapeutic agent.

The present invention provides a kit comprising:

-   -   a) a pharmaceutical formulation (e.g., one or more single dosage        forms) comprising a compound of the invention; and    -   b) instructions for the administration of the pharmaceutical        formulation, e.g., for treating or preventing any of the        conditions discussed above.

In certain embodiments, the kit further comprises instructions for theadministration of the pharmaceutical formulation comprising a compoundof the invention conjointly with a chemotherapeutic agent as mentionedabove. In certain embodiments, the kit further comprises a secondpharmaceutical formulation (e.g., as one or more single dosage forms)comprising a chemotherapeutic agent as mentioned above.

Definitions

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₆ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y)alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein each R¹⁰ independently represent a hydrogen or hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R¹⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group, or R⁹ and R¹⁰ taken together with theintervening atom(s) complete a heterocycle having from 4 to 8 atoms inthe ring structure.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3 to 8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl may be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl may be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR¹⁰ whereinR¹⁰ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl,alkenyl, alkynyl, or alkoxy substituents defined herein are respectivelylower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, orlower alkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl,such as alkyl, or R⁹ and R¹⁰ taken together with the intervening atom(s)complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—R¹⁰, wherein R¹⁰ represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR¹⁰ or—SC(O)R¹⁰ wherein R¹⁰ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl,such as alkyl, or either occurrence of R⁹ taken together with R¹⁰ andthe intervening atom(s) complete a heterocycle having from 4 to 8 atomsin the ring structure.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxylprotecting groups include,but are not limited to, those where the hydroxyl group is eitheracylated (esterified) or alkylated such as benzyl and trityl ethers, aswell as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol andpropylene glycol derivatives and allyl ethers.

The term “healthcare providers” refers to individuals or organizationsthat provide healthcare services to a person, community, etc. Examplesof “healthcare providers” include doctors, hospitals, continuing careretirement communities, skilled nursing facilities, subacute carefacilities, clinics, multispecialty clinics, freestanding ambulatorycenters, home health agencies, and HMO's.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention (e.g., a compound of formula I). Acommon method for making a prodrug is to include one or more selectedmoieties which are hydrolyzed under physiologic conditions to reveal thedesired molecule. In other embodiments, the prodrug is converted by anenzymatic activity of the host animal. For example, esters or carbonates(e.g., esters or carbonates of alcohols or carboxylic acids) arepreferred prodrugs of the present invention. In certain embodiments,some or all of the compounds of formula I in a formulation representedabove can be replaced with the corresponding suitable prodrug, e.g.,wherein a hydroxyl in the parent compound is presented as an ester or acarbonate or carboxylic acid present in the parent compound is presentedas an ester.

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In a preferred embodiment, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation ofpharmaceutical composition can be a selfemulsifying drug delivery systemor a selfmicroemulsifying drug delivery system. The pharmaceuticalcomposition (preparation) also can be a liposome or other polymermatrix, which can have incorporated therein, for example, a compound ofthe invention. Liposomes, for example, which comprise phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference. If desired, liquid ophthalmic formulations have propertiessimilar to that of lacrimal fluids, aqueous humor or vitreous humor orare compatable with such fluids. A preferred route of administration islocal administration (e.g., topical administration, such as eye drops,or administration via an implant).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the patient, which may includesynergistic effects of the two compounds). For example, the differenttherapeutic compounds can be administered either in the same formulationor in a separate formulation, either concomitantly or sequentially. Incertain embodiments, the different therapeutic compounds can beadministered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72hours, or a week of one another. Thus, an individual who receives suchtreatment can benefit from a combined effect of different therapeuticcompounds.

This invention includes the use of pharmaceutically acceptable salts ofcompounds of the invention in the compositions and methods of thepresent invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In certain embodiments, the invention relates to a method for conductinga pharmaceutical business, by manufacturing a formulation of a compoundof the invention, or a kit as described herein, and marketing tohealthcare providers the benefits of using the formulation or kit fortreating or preventing any of the diseases or conditions as describedherein.

In certain embodiments, the invention relates to a method for conductinga pharmaceutical business, by providing a distribution network forselling a formulation of a compound of the invention, or kit asdescribed herein, and providing instruction material to patients orphysicians for using the formulation for treating or preventing any ofthe diseases or conditions as described herein.

In certain embodiments, the invention comprises a method for conductinga pharmaceutical business, by determining an appropriate formulation anddosage of a compound of the invention for treating or preventing any ofthe diseases or conditions as described herein, conducting therapeuticprofiling of identified formulations for efficacy and toxicity inanimals, and providing a distribution network for selling an identifiedpreparation as having an acceptable therapeutic profile. In certainembodiments, the method further includes providing a sales group formarketing the preparation to healthcare providers.

In certain embodiments, the invention relates to a method for conductinga pharmaceutical business by determining an appropriate formulation anddosage of a compound of the invention for treating or preventing any ofthe disease or conditions as described herein, and licensing, to a thirdparty, the rights for further development and sale of the formulation.

EXAMPLES Example 1: Synthetic Protocols

To a suspension of 1019 (1.5 g, 6.8 mmol) in CH₂Cl₂ (15 mL) at 0° C. wasadded Et₃N (1.9 ml, 13.6 mmol) dropwise followed by phenyl acetylchloride (1.07 ml, 8.1 mmol) dropwise. The resulting mixture was stirredat 0° C. and then slowly warmed up to room temperature for 2 days. Thecrude material was purified by silica gel chromatography eluting with0-25% EtOAc in hexane to afford 1020.

To a solution of 4-bromo-1-butyne (7 g, 53 mmol) in DMSO (30 ml) at 0°C. was added NaI (7.94 g, 53 mmol). The mixture was stirred at roomtemperature for 2 h before it was cooled to 0° C. and followed byaddition of NaCN (5.2 g, 106 mmol). The resulting mixture was heated at80° C. for 2.5 h and then stirred at room temperature overnight. Themixture was partitioned between water and EtOAc. The organic extract waswashed with water, dried over sodium sulfate, filtered and evaporated toafford 1021.

To a mixture of 1020 (400 mg, 1.18 mmol), PdCl₂(PPh₃)₂(41 mg, 0.059mmol) and CuI (11 mg, 0.059 mmol) in Et₃N (3 ml) and THF (6 ml) underargon atmosphere was added 1021 (187 mg, 2.36 mmol), then heated at 60°C. overnight. After removal of the solvent, the residue was purified bysilica gel chromatography eluting with 0-60% EtOAc in Hexane to afford1022.

To a solution of 1022 (118 mg, 0.406 mmol) in the mixture of EtOAc (60ml) and EtOH (15 ml) was added Pd(OH)₂/C (50 mg, 0.356 mmol). Hydrogenwas bubbled through the resulting mixture and stirred for 1 h. The Pdcatalyst was filterd off and the filtrate was concentrated to afford1023.

A mixture of 1023 (127 mg, 0.431 mmol) and thiosemicarbazide (51 mg,0.561 mmol) in TFA (3 mL) was heated at 85° C. for 5 h. The reaction wascooled to room temperature and poured onto a mixture of ice-water. Themixture was basified with NaOH pellets (pH 10). The crude material waspurified by silica gel chromatography eluting with 0-6% MeOH in CH₂Cl₂to afford 1024.

To a solution of 1024 (38.4 mg, 0.104 mmol) in NMP (1 mL) at 0° C. wasadded phenyl acetyl chloride (0.017 mL, 0.125 mmol) dropwise. Theresulting mixture was stirred at 0° C. for 1.5 h before it was quenchedby addition of water (˜10 mL). The mixture was partitioned between waterand EtOAc. The organic extract was washed with water, dried over sodiumsulfate, filtered and evaporated. The crude material was purified bysilica gel chromatography eluting with 0-6% MeOH in CH₂Cl₂ to afford295. ¹H NMR (300 MHz, DMSO-d₆) δ 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19(d, J=8.82 Hz, 1H), 7.58-7.54 (d, J=9.72 Hz, 1H), 7.36-7.28 (m, 10H),3.81-3.78 (d, J=8.43 Hz, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs,4H).

Compound 1024 can also be prepared according to the following procedure:

To a solution of 3-amino-6-chloropyridazine (11.14 g, 86.0 mmol) in NMP(279 mL) at 19° C. was added phenylacetyl chloride (18.2 mL, 137.6 mmol)dropwise over 5 minutes with the internal temperature of the solutionmaintained T_(i)≤28° C. The resulting mixture was stirred at 19° C. for90 minutes and poured into ice water (557 mL). The white precipitate wascollected by suction filtration, rinsed with water (2×110 mL) anddiethyl ether (110 mL). The product was dried overnight under highvacuum to afford N-(6-chloropyridazin-3-yl)-2-phenylacetamide (xxx, 18.8g). ¹H NMR (300 MHz, DMSO-d₆) δ 11.57 (s, 1H), 8.40 (d, J=9.636 Hz, 1H),7.90 (d, J=9.516 Hz, 1H), 7.36 (m, 5H) 3.82 (s, 2H)

A 1000 mL three-neck flask fitted with internal temperature probe andaddition funnel was flushed with Ar_((g)). Under positive Argon pressure4-cyanobutylzinc bromide (0.5M in THF, 500 mL, 250 mmol) was chargedinto the addition funnel then added to the reaction vessel at roomtemperature. Solid N-(6-chloropyridazin-3-yl)-2-phenylacetamide (20.6 g,83.3 mmol) was added to the stirred solution at RT under Ar_((g)) flow,followed by the addition of NiCl₂(dppp) (4.52 g, 8.33 mmol). Theresulting mixture was stirred at 19° C. for 240 minutes and thenquenched with ethanol (120 mL). Water (380 mL) added to the stirred redsolution, giving a thick precipitate. Ethyl acetate (760 mL) added andstirred well for 30 minutes. The solids were removed by filtrationthrough a pad of celite. The mother liquor was then transferred to aseparatory funnel and the organic layer was washed with H₂O (380 mL),0.5% ethylenediaminetetraacetic acid solution (380 mL) and again withH₂O (380 mL). The organic layer was concentrated by rotoevaporation.Resulting red oil was redissolved in EtOAc (200 mL) and 1M HCl (380 mL)was added to the well stirred flask. After 30 minutes the mixture wastransferred to separatory funnel and the aqueous layer collected. Theorganic layer was extracted with 1M HCl (2×380 mL). The aqueous layer'spH was then adjusted to ˜7 using 7.5% sodium bicarbonate solution andthe pale yellow precipitate was collected by suction filtration, rinsedwith water (200 mL) and diethyl ether (2×200 mL). The solid was driedovernight under high vacuum to affordN-(6-(4-cyanobutyl)pyridazin-3-yl)-2-phenylacetamide (1023, 14.76 g). ¹HNMR (300 MHz, DMSO-d₆) δ 11.29 (s, 1H), 8.23 (d, J=9.036 Hz, 1H), 7.59(d, J=9.246 Hz, 1H), 7.32 (m, 5H), 3.79 (s, 2H), 2.90 (t, J=7.357 Hz,2H), 2.56 (t, J=7.038 Hz, 2H), 1.79 (t, J=7.311 Hz, 2H), 1.63 (t, J=7.01Hz, 2H)

N-(6-(4-cyanobutyl)pyridazin-3-yl)-2-phenylacetamide (14.7 g, 50.2 mmol)was charged into a 250 mL round bottom flask fitted with an open topreflux condenser. To the flask was added thiosemicarbazide (5.03 g, 55.2mmol) and trifluoroacetic acid (88 mL). The reaction slurry was heatedin a 65° C. bath for 2 h. After cooling to RT, H₂O (150 mL) was addedand stirred for 30 minutes. The mixture was then slowly transferred to astirred 7.5% sodium bicarbonate solution (1400 mL) cooled in a 0° C.bath. The precipitate was collected by suction filtration, rinsed withwater (2×200 mL), diethyl ether (2×200 mL) and dried under high vacuumovernight. The off-white solid was slurried in DMSO (200 mL) and heatedin an 80° C. bath until the internal temperature reached 65° C. DMSO(105 mL) was used to rinse sides of flask. H₂O (120 mL) was slowly addeduntil the solution became slightly cloudy and then the mixture wasremoved from heat bath and allowed to cool to ambient temperature whilestirring. The pale green precipitate was collected by suctionfiltration, rinsed with water (200 mL) and diethyl ether (2×200 mL). Thesolid was dried overnight under high vacuum to provideN-(6-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)-2-phenylacetamide(1024, 15.01 g). ¹H NMR (300 MHz, DMSO-d₆) δ 11.28 (s, 1H), 8.23 (d,J=8.916 Hz, 1H), 7.59 (d, J=8.826 Hz, 1H), 7.36 (m, 5H), 7.07 (s, 2H),3.78 (s, 2H), 2.87 (t, J=6.799 Hz, 4H), 1.69 (bm, 4H).

A flask was charged with 1024 (500 mg, 1.36 mmol), DL-mandelic acid (248mg, 1.63 mmol) in DMF (10 ml) at 0° C. was added HOBT (441 mg, 3.26mmol) followed by EDCI (781 mg, 4.08 mmol). The resulting mixture wasstirred at 0° C. for 10 minutes then warmed up to room temperature andstirred for 10 minutes before it was quenched by addition of water (˜50mL) at 0° C. The white precipitate was collected by suction filtration,rinsed with more water and dried to afford 315. ¹H NMR (300 MHz,DMSO-d₆) δ 12.65 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H),7.58-7.50 (m, 3H), 7.36-7.28 (m, 8H), 6.35 (s, 1H), 5.32 (s, 1H), 3.78(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

To a suspension of 3-morpholin-4-yl-propionic acid hydrochloride (209mg, 1.07 mmol) in DMF (10 ml) was added EDCI (308 mg, 1.61 mmol). Theresulting mixture was stirred at 0° C. for 1 hour and followed byaddition of 315 (447 mg, 0.889 mmol) and 4-DMAP (261 mg, 2.14 mmol). Theresulting mixture was stirred from 0° C. to room temperature over aperiod of 6 h before it was quenched by addition of ice water (˜50 mL).The white precipitate was collected by suction filtration, rinsed withmore water. The crude material was purified by silica gel chromatographyeluting with 0-6% MeOH in EtOAc to afford 334. ¹H NMR (300 MHz, DMSO-d₆)δ 12.95 (s, 1H), 11.26 (s, 1H), 8.22-8.19 (d, J=9.45 Hz, 1H), 7.58-7.26(m, 11H), 6.14 (s, 1H), 3.78 (s, 2H), 3.54 (bs, 4H), 3.01 (bs, 2H), 2.90(bs, 2H), 2.63 (bs, 4H), 2.38 (bs, 4H), 1.73 (bs, 4H).

A flask was charged with 1024 (50 mg, 0.135 mmol), 3-chlorophenylaceticacid (28 mg, 0.163 mmol) in DMF (1 ml) at 0° C. was added HOBT (44 mg,0.326 mmol) followed by EDCI (78 mg, 0.408 mmol). The resulting mixturewas slowly warmed up to room temperature and stirred for 1 h before itwas quenched by addition of water (˜5 mL). The white precipitate wascollected by suction filtration, rinsed with more water and ether thendried to afford 335. ¹H NMR (300 MHz, DMSO-d₆) δ 12.65 (s, 1H), 11.26(s, 1H), 8.22-8.19 (d, J=8.82 Hz, 1H), 7.58-7.54 (d, J=9.72 Hz, 1H),7.36-7.28 (m, 9H), 3.84 (s, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs,2H), 1.73 (bs, 4H).

Compound 413 was prepared according to the procedure above for thepreparation of compound 315. ¹H NMR (300 MHz, DMSO-d₆) δ 12.68 (bs, 1H),11.26 (s, 1H), 8.20 (d, J=9.46 Hz, 1H), 7.58-7.26 (m, 10H), 3.90 (s,2H), 3.78 (s, 2H), 3.02 (bs, 2H), 2.90 (bs, 2H), 1.74 (bs, 4H).

To a suspension of 295 (30 mg, 0.0617 mmol) in MeOH (2 ml) at 0° C. wasadded 2N NaOH (2 ml) solution. The resulting mixture was stirred at roomtemperature overnight. The solvent was evaporated under vacuo and themixture was acidified with 1N HCl to pH 6. The white precipitate wascollected by suction filtration, rinsed with more water and dried toafford 348. ¹H NMR (300 MHz, DMSO-d₆) δ 7.32-7.24 (m, 5H), 7.15-7.12 (d,J=9.57 Hz, 1H), 6.72-6.69 (d, J=9.15 Hz, 1H), 6.09 (s, 2H), 3.77 (s,2H), 2.99-2.96 (bs, 2H), 2.76-2.70 (bs, 2H), 1.70 (bs, 4H).

To a mixture of 413 (1.62 g) in MeOH (25 mL), THF (10 mL) and H₂O (10mL) at room temperature was added 1N aq. NaOH (8 mL). This mixture wasstirred for 24 h before the organic volatile was removed under reducedpressure. The residue was neutralized to pH 7 with 1N aq. HCl solutionand extracted with EtOAc (2×20 mL). The combined extract was dried(MgSO₄) and concentrated. The crude was purified by silica gelchromatography eluting with 1-15% MeOH in dichloromethane (DCM) toafford amine 1116. The resulting amine 1116 was converted to 660 asdescribed for 335. ¹H NMR (300 MHz, DMSO-d₆) δ 12.68 (bs, 1H), 11.31 (s,1H), 8.20 (d, J=9.2 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.52-7.21 (m, 8H),3.90 (s, 2H), 3.87 (s, 2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

3-Amino-6-chloropyridazine (55.5 g, 0.428 mol) and3-(Trifluoromethoxy)phenylacetic acid (1.1 equiv., 0.471 mol, 104 g)were dissolved in DMF (30.0 vol., 1.66 L) in a 3000 mL three neckround-bottom flask. Addition of DIEA (1.1 equiv., 0.471 mol, 82 mL) viaaddition funnel was done over 5 minutes. Propylphosphonic anhydridesolution (300 mL of a 50% solution in DMF, 1.1 equiv., 0.471 mol) wascharged into a 500 mL addition funnel and added dropwise to reactionsolution (keeping reaction temperature ≤+30° C.). The reaction usuallygoes to completion after 3 hours (TLC: 6:4 hexanes-ethyl acetate).Reaction mixture was then poured into 7.5% sodium bicarbonate (80.0vol., 4.4 L) which was chilled in an ice bath. Off-white crystallinepowder was filtered through a Büchner funnel, rinsed with water (20.0vol., 1.1 L). Dried in a 50° C. vacuum to a constant weight to affordN-(6-chloropyridazin-3-yl)-2-β-(trifluoromethoxy)phenyl)acetamide 1117:yield of 119.6 g (77%). ¹H NMR (300 MHz, DMSO-d₆) δ 11.63 (s, 1H), 8.38(d, J=9.4 Hz, 1H), 7.88 (d, J=9.4 Hz, 1H), 7.52-7.27 (m, 4H), 3.90 (s,2H).

4-Cyanobutylzinc bromide solution (3.0 equiv., 0.50 mol, 1.0 L) wascharged into an argon gas purged 5000 mL 3 neck round bottom flask.Argon_((g)) purge for 5 minutes followed by the addition of 1117 (1.0equiv., 0.167 mol, 55.3 g) and NiCl₂(dppp) (0.15 equiv., 0.0251 mol,13.6 g) under a blanket of argon_((g)). The reaction usually goes tocompletion after 4 hours (TLC: 1:1 hexanes-ethyl acetate). EtOAc (15vol., 832 mL) added to deep red solution. Water (15 vol., 832 mL) wasadded, thick slurry formed. 1N HCl added until slurry breaks to paleblue layer (˜6 vol., 333 mL). Transferred to separatory funnel andorganic layer was washed with 1N HCl (2×500 mL), dried (MgSO₄) andconcentrated by rotary evaporation (bath ≤30° C.) to a solid reddishoil. Oil dissolved in dichloromethane (15 vol., 832 mL), silica gel (100g) was slurried into red solution, this was concentrated by rotaryevaporation (bath ≤30° C.) to a solid reddish powder. Loaded onto a bedof silica gel (5 cm×11 cm), flushed with 25% hexanes in ethyl Acetate (3L), combined organics concentrated by rotary evaporation (bath ≤30° C.).Dried under high vacuum to a constant weight to affordN-(6-(4-cyanobutyl)pyridazin-3-yl)-2-β-(trifluoromethoxy)phenyl)acetamide1118: yield of 58.2 g (92%). ¹H NMR (300 MHz, DMSO-d₆) δ 11.41 (s, 1H),8.28 (d, J=9.2 Hz, 1H), 7.65 (d, J=9.2 Hz, 1H), 7.52-7.27 (m, 4H), 3.89(s, 2H), 2.92 (t, J=7.5 Hz, 2H), 2.56 (t, J=7.0 Hz, 2H), 1.80 (m, 2H),1.61 (m, 2H).

1118 (1.0 equiv., 0.154 mol, 58.2 g) was charged into a 500 mL roundbottom flask along with thiosemicarbazide (1.2 equiv., 0.184 mol, 16.8g). TFA (5 vol., 291 mL) slowly added to reaction vessel while stirring.The reaction slurry was heated in a 65° C. bath with an open top refluxcondenser. The reaction usually goes to completion after 5 hours(determined by LC/MS). Toluene (10 vol., 582 mL) added to deep redsolution, azeotroped by rotary evaporation (bath ≤30° C.) to a red oil.Slowly transferred oil to a well stirred 6000 mL Erlenmeyer flaskcontaining 7.5% sodium bicarbonate solution (69 vol., 4.0 L) cooled in a0° C. bath. The crystals were filtered through a Büchner funnel andrinsed twice with diethyl ether (5 vol., 2×250 mL). Dried under highvacuum to a constant weight to affordN-(6-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)-2-β-(trifluoromethoxy)phenyl)acetamide657; yield of 55.7 g (80%). ¹H NMR (300 MHz, DMSO-d₆) δ 11.33 (s, 1H),8.21 (d, J=9.2 Hz, 1H), 7.58 (d, J=9.2 Hz, 1H), 7.51-7.26 (m, 4H), 6.99(s, 2H), 3.88 (s, 2H), 2.87 (m, 4H), 1.71 (m, 4H).

To a solution of 657 (50 mg, 0.11 mmol) in DMF (3 mL) at 0° C. was added4-fluorophenyl acetic acid (22 mg, 0.14 mmol), HOBt (30 mg, 0.22 mmol)and EDCI (42 mg, 0.22 mmol). The resulting mixture was stirred at roomtemperature for 1.5 h before it was cooled to 0° C. and quenched withH₂O. The precipitate was collected by suction filtration and furtherpurified by silica gel chromatography eluting with 1-10% MeOH in DCM toafford 661. ¹H NMR (300 MHz, DMSO-d₆) δ 12.65 (bs, 1H), 11.31 (s, 1H),8.20 (d, J=9.1 Hz, 1H), 7.57 (d, J=9.4 Hz, 1H), 7.49-7.14 (m, 8H), 3.87(s, 2H), 3.81 (s, 2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

662 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.67 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.1 Hz,1H), 7.57 (d, J=9.1 Hz, 1H), 7.51-7.07 (m, 7H), 3.89 (s, 2H), 3.87 (s,2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

663 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.74 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.2 Hz,1H), 7.57 (d, J=9.2 Hz, 1H), 7.51-7.19 (m, 7H), 3.97 (s, 2H), 3.87 (s,2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

To a mixture of 1-bromo-3-(difluoromethoxy)benzene (1 g, 4.5 mmol),bis(tri-tert-butylphosphine) palladium(0) (460 mg, 0.9 mmol) in1,4-dioxane (30 ml) under argon atmosphere was added 0.5 M of2-tert-butoxy-2-oxoethyl zinc chloride in ether (22.5 ml). The resultingmixture was stirred at room temperature overnight. The mixture waspartitioned between saturated NH₄Cl and EtOAc. The organic extract waswashed with brine, dried over sodium sulfate, filtered and evaporated.The crude material was purified by silica gel chromatography elutingwith 0-10% EtOAc in Hexane to afford 1119.

To a solution of 1119 (300 mg, 1.16 mmol) in DCM (5 ml) at 0° C. wasadded TFA (3 ml) dropwise. The resulting mixture was stirred at roomtemperature overnight before it was evaporated to dryness thentriturated the residue with ether to afford 1120.

A flask was charged with 348 (50 mg, 0.135 mmol), 1120 (28 mg, 0.142mmol) in DMF (1 ml) at 0° C. was added HOBT (39 mg, 0.285 mmol) followedby EDCI (68 mg, 0.356 mmol). The resulting mixture was slowly warmed upto room temperature and stirred overnight before it was quenched byaddition of ice water (˜5 mL). The white precipitate was collected bysuction filtration, rinsed with more water. The crude material waspurified by silica gel chromatography eluting with 0-6% MeOH in DCM toafford 666. ¹H NMR (300 MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.32 (s, 1H),8.22-8.19 (d, J=9.12 Hz, 1H), 7.58-7.54 (d, J=9.03 Hz, 1H), 7.48-6.98(m, 10H), 3.81 (bs, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

668 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.15 Hz,1H), 7.58-6.99 (m, 10H), 3.87-3.84 (d, 4H), 3.01 (bs, 2H), 2.90 (bs,2H), 1.73 (bs, 4H).

669 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.09 Hz,1H), 7.58-7.54 (d, J=9.37 Hz, 1H), 7.48-7.28 (m, 6H), 7.03-6.97 (m, 2H),4.77-4.74 (q, 2H), 3.87 (s, 2H), 3.78 (s, 2H), 3.01 (bs, 2H), 2.90 (bs,2H), 1.73 (bs, 4H).

A flask was charged with 657 (50 mg, 0.111 mmol), 2-pyridine acetic acidhydrochloride (20 mg, 0.116 mmol) in DMF (1 ml) at 0° C. was treatedwith propylphosphonic anhydride solution (91 ul) followed bytriethylamine (40 ul, 0.29 mmol). The resulting mixture was slowlywarmed up to room temperature and stirred for 1 h before it was quenchedby addition of ice water (˜5 mL). The yellow precipitate was collectedby suction filtration, rinsed with more water. The crude material waspurified by silica gel chromatography eluting with 0-6% MeOH in DCM toafford 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.67 (s, 1H), 11.32 (s, 1H),8.53-8.49 (m, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.78-7.76 (t, 1H),7.58-7.26 (m, 7H), 4.01 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs,2H), 1.73 (bs, 4H).

671 was made using procedure described for compound 670. ¹H NMR (300MHz, DMSO-d₆) δ 12.70 (s, 1H), 11.32 (s, 1H), 8.53-8.48 (m, 2H),8.22-8.19 (d, J=9.12 Hz, 1H), 7.76-7.26 (m, 7H), 3.87 (s, 4H), 3.01 (bs,2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

672 was made using procedure described for compound 670. ¹H NMR (300MHz, DMSO-d₆) δ 11.32 (s, 1H), 8.53-8.52 (bs, 2H), 8.22-8.19 (d, J=9.12Hz, 1H), 7.58-7.26 (m, 7H), 3.87 (s, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H),1.73 (bs, 4H).

673 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.69 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.1 Hz,1H), 7.57 (d, J=9.1 Hz, 1H), 7.51-7.21 (m, 8H), 3.90 (s, 2H), 3.87 (s,2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

674 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.63 (bs, 1H), 11.32 (s, 1H), 8.20 (d, J=9.2 Hz,1H), 7.57 (d, J=9.2 Hz, 1H), 7.51-7.38 (m, 3H), 7.33-7.09 (m, 5H), 3.87(s, 2H), 3.79 (s, 2H), 3.06-2.86 (m, 4H), 2.48 (s, 3H), 1.77-1.72 (m,4H).

A flask was charged with 657 (70 mg, 0.155 mmol), 5-pyrimidineaceticacid (22 mg, 0.162 mmol) in DMF (1 ml) at 0° C. was added HOBT (44 mg,0.326 mmol) followed by EDCI (78 mg, 0.408 mmol). The resulting mixturewas slowly warmed up to room temperature and stirred for overnightbefore it was quenched by addition of ice water (˜5 mL). The whiteprecipitate was collected by suction filtration, rinsed with more water.The crude material was purified by silica gel chromatography elutingwith 0-6% MeOH in DCM to afford 675. ¹H NMR (300 MHz, DMSO-d₆) δ 12.75(s, 1H), 11.32 (s, 1H), 9.11 (s, 1H), 8.76 (s, 1H), 8.22-8.19 (d, J=9.12Hz, 1H), 7.59-7.26 (m, 6H), 3.94 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H),2.90 (bs, 2H), 1.73 (bs, 4H).

676 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.70 (s, 1H), 8.61-8.57(m, 2H), 8.22-8.19 (d, J=9.36 Hz, 1H), 7.59-7.26 (m, 5H), 4.11 (s, 2H),3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

677 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.89 (s, 1H), 8.22-8.19(d, J=9.15 Hz, 1H), 7.59-7.26 (m, 5H), 6.62 (s, 1H), 3.99 (s, 2H), 3.87(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

678 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 9.06 (s, 1H), 8.22-8.19(d, J=9.21 Hz, 1H), 7.59-7.26 (m, 6H), 4.03 (s, 2H), 3.87 (s, 2H), 3.01(bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

679 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.67 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.2 Hz,1H), 7.57 (d, J=9.2 Hz, 1H), 7.51-7.36 (m, 4H), 7.29-7.12 (m, 4H), 3.87(s, 2H), 3.85 (s, 2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

680 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.67 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.3 Hz,1H), 7.57 (d, J=9.0 Hz, 1H), 7.51-7.28 (m, 8H), 3.87 (s, 2H), 3.84 (s,2H), 3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

To a solution of 674 (100 mg, 0.16 mmol) in DCM at −78° C. was addedm-CPBA (60 mg, 0.24 mmol) in 4 portions. The resulting mixture wasstirred at that temperature for 1 h before it was slowly warmed up to−10° C. and quenched with 25% aq. Na₂S₂O₃ solution. The reaction wasdiluted with EtOAc, washed with saturated aq. NaHCO₃ (3×10 mL). Thecombined organic layer was separated, washed with brine, dried (MgSO₄)and concentrated. The crude was purified by HPLC to afford 682. ¹H NMR(300 MHz, DMSO-d₆) δ 12.72 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.0 Hz,1H), 7.68 (m, 1H), 7.60-7.26 (m, 8H), 3.91 (s, 2H), 3.87 (s, 2H),3.06-2.86 (m, 4H), 2.76 (s, 3H), 1.77-1.72 (m, 4H).

681 was prepared from 657 and 3-methylsulphonylphenyl acetic acid by theprocedure as described for compound 661. ¹H NMR (300 MHz, DMSO-d₆) δ12.72 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.0 Hz, 1H), 7.92-7.83 (m,2H), 7.70-7.26 (m, 7H), 3.93 (s, 2H), 3.87 (s, 2H), 3.23 (s, 3H),3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

683 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.36 (s, 1H), 8.21-8.18(d, J=9.18 Hz, 1H), 7.84-7.80 (d, J=9.36 Hz, 1H), 7.59-7.26 (m, 6H),3.90-3.87 (d, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

684 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.57 (s, 1H), 8.51-8.49(d, J=9.18 Hz, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.79-7.75 (d, J=9.36Hz, 1H), 7.59-7.26 (m, 6H), 4.07 (t, 2H), 3.87 (s, 2H), 3.30-3.28 (m,1H), 3.19 (s, 3H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.3-2.5 (m, 1H),1.99-1.96 (m, 1H), 1.73 (bs, 4H).

685 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.52 (bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.1 Hz,1H), 7.61-7.25 (m, 7H), 3.87 (s, 2H), 3.80 (s, 3H), 3.62 (s, 2H),3.06-2.86 (m, 4H), 1.77-1.72 (m, 4H).

686 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.53 (bs, 1H), 11.32 (s, 1H), 8.20 (d, J=9.1 Hz,1H), 7.58 (d, J=9.2 Hz, 1H), 7.52-7.26 (m, 4H), 5.96 (s, 1H), 3.87 (s,2H), 3.67 (s, 2H), 3.64 (s, 3H), 3.06-2.86 (m, 4H), 2.21 (s, 3H),1.77-1.72 (m, 4H).

687 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.56 (bs, 1H), 11.32 (s, 1H), 8.20 (d, J=9.3 Hz,1H), 7.61-7.38 (m, 6H), 6.17 (d, J=2.2 Hz, 1H), 3.87 (s, 2H), 3.79 (s,3H), 3.75 (s, 2H), 3.03-2.90 (m, 4H), 1.7-1.72 (m, 4H).

688 was prepared by the procedure as described for compound 661. ¹H NMR(300 MHz, DMSO-d₆) δ 12.61 (bs, 1H), 11.32 (s, 1H), 8.20 (d, J=9.3 Hz,1H), 7.58 (d, J=9.3 Hz, 1H), 7.51-7.26 (m, 4H), 3.87 (s, 2H), 3.84 (s,2H), 3.07-2.86 (m, 4H), 1.77-1.72 (m, 4H).

To a solution of 657 (200 mg, 0.44 mmol) in DMF (4 mL) at 0° C. wasadded mandelic acid (124 mg, 0.66 mmol), HOBt (119 mg, 0.88 mmol) andEDCI (170 mg, 0.88 mmol). The resulting mixture was stirred at roomtemperature for 1.5 h before it was cooled to 0° C. and quenched withH₂O. The precipitate was collected by suction filtration and furtherpurified by silica gel chromatography eluting with 1-10% MeOH in DCM toafford 690 and a more polar 689. 689: ¹H NMR (300 MHz, DMSO-d₆) δ 12.42(bs, 1H), 11.31 (s, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.58-7.27 (m, 10H),6.35 (d, J=4.4 Hz, 1H), 5.34 (d, J=4.3 Hz, 1H), 3.87 (s, 2H), 3.03-2.89(m, 4H), 1.77-1.73 (m, 4H). 690: ¹H NMR (300 MHz, DMSO-d₆) δ 13.05 (bs,1H), 11.31 (s, 1H), 8.20 (d, J=9.0 Hz, 1H), 7.59-7.26 (m, 15H), 6.26 (d,J=5.5 Hz, 1H), 6.11 (s, 1H), 5.38 (d, J=5.3 Hz, 1H), 3.87 (s, 2H),3.03-2.88 (m, 4H), 1.76-1.73 (m, 4H).

447 was prepared from 657 and 3-chloromandelic acid by the procedure asdescribed for compound 689. ¹H NMR (300 MHz, DMSO-d₆) δ 12.48 (bs, 1H),11.31 (s, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.59-7.26 (m, 9H), 6.53 (m, 1H),5.36 (t, J=0.7 Hz, 1H), 3.87 (s, 2H), 3.03-2.90 (m, 4H), 1.75-1.71 (m,4H).

692 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.21-8.18 (d, J=9.18 Hz,1H), 7.80-7.26 (m, 9H), 3.92 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90(bs, 2H), 1.73 (bs, 4H).

693 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.75 (s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.06 Hz,1H), 7.79 (s, 1H), 7.59-7.26 (m, 6H), 6.31 (s, 1H), 5.20 (s, 2H), 3.87(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

694 was made using procedure described for compound 675. ¹H NMR (300MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.32 (s, 1H), 8.22-8.18 (d, J=9.15 Hz,1H), 7.58-7.54 (d, J=9.18 Hz, 1H), 7.48-7.26 (m, 4H), 3.87 (s, 2H), 3.63(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.39 (s, 3H), 2.13 (s, 3H), 1.73(bs, 4H), 1.57 (s, 9H).

To a solution of 694 (50 mg, 0.081 mmol) in DCM (2 ml) was added TFA (2ml) at 0° C. The resulting mixture was stirred at room temperature for 1h before it was evaporated under vacuo to dryness. Ether was added andthe white precipitate was collected by suction filtration, rinsed withmore ether to afford 695. ¹H NMR (300 MHz, DMSO-d₆) δ 12.71 (s, 1H),11.32 (s, 1H), 8.22-8.19 (d, J=9.36 Hz, 1H), 7.60-7.57 (d, J=9.27 Hz,1H), 7.51-7.28 (m, 4H), 3.88 (s, 2H), 3.57 (s, 2H), 3.01 (bs, 2H), 2.90(bs, 2H), 2.45 (s, 3H), 2.15 (s, 3H), 1.73 (bs, 4H).

696 was made using procedure described for compound 695. ¹H NMR (300MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.30 Hz,1H), 8.15 (s, 1H), 7.58-7.54 (d, J=9.30 Hz, 1H), 7.48-7.28 (m, 5H), 3.87(s, 2H), 3.76 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H), 1.59(s, 9H).

697 was made using procedure described for compound 695. ¹H NMR (300MHz, DMSO-d₆) δ 14.22 (s, 1H), 12.71 (s, 1H), 11.32 (s, 1H), 9.01 (s,1H), 8.22-8.19 (d, J=9.15 Hz, 1H), 7.59-7.26 (m, 6H), 4.04 (s, 2H), 3.87(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.73 (bs, 4H).

To a suspension of 3-morpholin-4-yl-propionic acid hydrochloride (113mg, 0.58 mmol) in DMF (8 mL) at 0° C. was addedN-β-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (130 mg,0.67 mmol). The resulting mixture was stirred at 0° C. for 40 min andfollowed by addition of 689 (300 mg, 0.48 mmol) and 4-DMAP (165 mg, 1.35mmol). The resulting mixture was stirred from 0° C. to room temperatureover a period of 3.5 h before it was diluted with EtOAc and cold water.The organic layer was separated and washed with water (3×15 mL), brine,dried (MgSO₄) and concentrated. The crude product was purified by silicagel chromatography eluting with 0-15% MeOH in CH₂Cl₂ to provide 711 (297mg) as white solid. ¹H NMR (300 MHz, CDCl₃) δ 10.75 (bs, 1H), 8.49 (d,J=9.0 Hz, 1H), 7.64 (s, 1H), 7.50-7.26 (m, 7H), 7.16-7.15 (m, 1H), 6.51(s, 1H), 4.04 (s, 2H), 3.80-3.72 (m, 4H), 3.88-2.81 (m, 8H), 2.75-2.71(m, 5H), 1.89 (m, 4H).

A mixture of 1117 (4.00 g, 12.06 mmol), 4-pentynenitrile (2.11 mL, 24.12mmol), PdCl₂(PPh₃)₂ (847 mg, 1.21 mmol), CuI (184 mg, 0.96 mmol) andEt₃N (13.44 mL, 96.48 mmoL) in DMF (18 mL) was heated at 55° C. for 5 h.The reaction was cooled to room temperature and poured into a mixture ofice-water. The precipitate was collected by suction filtration and airdried. The crude product was further recrystallized from a mixture ofi-PrOH—H₂O first and then from i-PrOH to provide alkyne 1131.

A mixture of alkyne 1131 (6.00 g) and Pd(OH)₂/C (1.00 g) in a mixture ofEtOAc (150 mL), THF (75 mL) and MeOH (75 mL) was stirred under 1 atm ofD₂ at room temperature for 3 h before the catalyst was filtered off ashort plug of SiO₂ and rinsed with EtOAc. The filtrate was concentratedto provide the crude product which was further recrystallized from amixture of EtOAc and ether to give the desired alkane 1132 as off-whitesolid (6.01 g)

A mixture of nitrile 1132 (5.20 g, 13.61 mmol) and thiosemicarbazide(1.61 g, 17.69 mmol) in TFA (75 mL) was heated at 80° C. for 4 h. Thereaction was cooled to room temperature and poured into a mixture ofice-water. The mixture was basified with NaOH pellets (pH 14). The whiteprecipitate was collected by suction filtration, rinsed with water anddried to provide 726 (5.87 g).

To a solution of 726 (1.40 g, 3.07 mmol) and 2-pyridylacetic acid HClsalt (1.49 g, 8.59 mmol) in DMF (20 mL) at 0° C. was added Et₃N (1.50mL, 10.73 mmol) and followed by 1-propanephosphonic anhydride (2.73 mL,50% in DMF, 4.29 mmol). This mixture was stirred for 2.5 h at roomtemperature before it was cooled back to 0° C. and quenched withice-H₂O. The precipitate was collected by suction filtration and airdried. This crude product was further purified by silica gelchromatography eluting with 0-15% MeOH in DCM to afford 727 (0.97 g). ¹HNMR (300 MHz, DMSO-d₆) δ 12.67 (s, 1H), 11.31 (s, 1H), 8.52-8.50 (m,1H), 8.20 (d, J=9.2 Hz, 1H), 7.78 (dt, J=1.8, 7.6 Hz, 1H), 7.58 (d,J=9.1 Hz, 1H), 7.51-7.26 (m, 6H), 4.02 (s, 2H), 3.87 (s, 2H), 3.03 (t,J=7.4 Hz, 2H), 1.73 (t, J=7.4 Hz, 2H).

Compound 710 was prepared from compound 447 using a procedure analogousto that employed for the preparation of compound 711. ¹H NMR (300 MHz,DMSO-d₆) δ 11.32 (s, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.62-7.26 (m,9H), 6.16 (s, 1H), 3.87 (s, 2H), 3.52-3.50 (d, 2H), 3.01 (bs, 2H), 2.90(bs, 2H), 2.80-2.71 (m, 11H), 1.73 (bs, 4H).

Compound 712 was prepared from compound 447 using a procedure analogousto that employed for the preparation of compound 711. ¹H NMR (300 MHz,DMSO-d₆) δ 11.32 (s, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.62-7.26 (m,9H), 6.16 (s, 1H), 3.87 (s, 2H), 3.38-3.36 (d, 2H), 3.01 (bs, 2H), 2.90(bs, 2H), 2.29 (s, 6H), 1.73 (bs, 4H).

Compound 713 was prepared from compound 447 using a procedure analogousto that employed for the preparation of compound 711. ¹H NMR (300 MHz,DMSO-d₆) δ 13.11 (bs, 1H), 11.32 (s, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H),7.62-7.26 (m, 9H), 6.16 (s, 1H), 3.87 (s, 2H), 3.60-3.57 (m, 4H),3.44-3.42 (d, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.55-2.51 (m, 4H), 1.73(bs, 4H).

Compound 714 was prepared from compound 447 using a procedure analogousto that employed for the preparation of compound 711. ¹H NMR (300 MHz,DMSO-d₆) δ 11.32 (s, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.62-7.26 (m,9H), 6.16 (s, 1H), 3.87 (s, 2H), 3.38-3.31 (d, 2H), 3.01 (bs, 2H), 2.90(bs, 2H), 2.49-2.47 (m, 4H), 1.93 (bs, 4H), 1.73 (bs, 4H), 1.72 (bs,2H).

To a suspension of 670 (3 g, 5.24 mmol) in MeOH (50 ml) at 0° C. wasadded 2N NaOH (20 ml) solution. The resulting mixture was stirred atroom temperature overnight. The solvent was evaporated under vacuo andthe mixture was acidified with 1N HCl to pH 6. The white precipitate wascollected by suction filtration, rinsed with more water and dried toafford 1121a. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66 (s, 1H), 8.51-8.50 (m,1H), 7.81-7.76 (m, 1H), 7.42-7.28 (m, 2H), 7.16-7.13 (d, 1H), 6.73-6.70(d, 1H), 6.10 (s, 2H), 4.0 (s, 2H), 3.01 (bs, 2H), 2.71 (bs, 2H), 1.70(bs, 4H).

To a solution of 1121a (20 mg, 0.054 mmol) in DMF (1 ml) at 0° C. wasadded triethylamine (11 ul, 0.081 mmol) drop wise followed byo-acetylmandelic acid chloride (15 ul, 0.065 mmol) drop wise. Theresulting mixture was slowly warmed up to room temperature and stirredfor 1 h before it was quenched by addition of water (˜3 mL) at 0° C. Themixture was partitioned between water and EtOAc. The organic extract waswashed with brine, dried over sodium sulfate, filtered and evaporated.The crude material was purified by silica gel chromatography elutingwith 0-5% MeOH in DCM to afford 1122.

A flask was charged with 1122 (20 mg, 0.037 mmol) and 2N ammonia in MeOH(5 ml). The mixture was stirred at room temperature for 2 hours. Thesolvent was evaporated under vacuo and the mixture was triturated withether. The white precipitate was collected by suction filtration, rinsedwith ether and dried to afford 715. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66(s, 1H), 10.61 (s, 1H), 8.51-8.50 (m, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H),7.81-7.76 (m, 1H), 7.61-7.53 (m, 3H), 7.42-7.28 (m, 5H), 6.49-6.47 (d,1H), 5.30-5.28 (d, 1H), 4.0 (s, 2H), 3.02 (bs, 2H), 2.91 (bs, 2H), 1.75(bs, 4H).

Compound 719 was prepared using a procedure analogous to that employedfor the preparation of compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66(s, 1H), 11.32 (s, 1H), 8.51-8.50 (m, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H),7.79-7.76 (m, 1H), 7.59-7.30 (m, 6H), 4.0 (s, 2H), 3.87 (s, 2H), 3.01(bs, 2H), 2.90 (bs, 2H), 1.75 (bs, 4H).

Compound 720 was prepared using a procedure analogous to that employedfor the preparation of compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66(s, 1H), 11.32 (s, 1H), 8.51-8.50 (m, 1H), 8.19-8.16 (d, J=9.06 Hz, 1H),7.79-7.76 (m, 1H), 7.59-7.30 (m, 6H), 4.01 (s, 2H), 3.95 (s, 2H), 3.03(bs, 2H), 2.91 (bs, 2H), 1.76 (bs, 4H).

Compound 721 was prepared using a procedure analogous to that employedfor the preparation of compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66(s, 1H), 11.32 (s, 1H), 8.51-8.50 (m, 1H), 8.21-8.16 (d, J=9.06 Hz, 1H),7.81-7.28 (m, 7H), 4.01 (s, 2H), 3.89 (s, 2H), 3.03 (bs, 2H), 2.91 (bs,2H), 1.76 (bs, 4H).

Compound 717 was prepared using a procedure analogous to that employedfor the preparation of compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66(s, 1H), 11.17 (s, 1H), 8.52-8.50 (m, 1H), 8.19-8.16 (d, J=9.06 Hz, 1H),7.81-7.76 (m, 1H), 7.58-7.55 (d, 1H), 7.42-7.09 (m, 4H), 7.08-7.06 (d,1H), 4.01 (s, 2H), 3.83 (s, 2H), 3.79 (s, 3H), 3.03 (bs, 2H), 2.91 (bs,2H), 1.76 (bs, 4H).

To a solution of 717 (10 mg, 0.017 mmol) in DCM (3 ml) at 0° C. wasadded boron tribromide solution (1N in DCM) (2 ml) drop wise. Theresulting mixture was slowly warmed up to room temperature and stirredfor 4.5 h before it was quenched by addition of water (˜3 mL). Themixture was then basified with 1N NaOH to pH 8. The mixture waspartitioned between water and DCM. The organic extract was washed withbrine, dried over sodium sulfate, filtered and evaporated. The crudematerial was purified by silica gel chromatography eluting with 0-10%MeOH in DCM to afford 718. ¹H NMR (300 MHz, DMSO-d₆) δ 11.17 (s, 1H),8.52-8.50 (m, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.81-7.76 (m, 1H),7.58-7.55 (d, 1H), 7.51-7.09 (m, 4H), 6.88-6.85 (d, 1H), 4.0 (s, 2H),3.79 (s, 2H), 3.03 (bs, 2H), 2.91 (bs, 2H), 1.76 (bs, 4H).

Compound 1128 was prepared from 4-bromo-2-trifluoromethoxyanisole usinga procedure analogous to that for compound 1124 below.

Compound 722 was prepared using compound 1128 with a procedure analogousto that for compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66 (s, 1H),11.17 (s, 1H), 8.52-8.50 (m, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H),7.81-7.76 (m, 1H), 7.58-7.55 (d, 1H), 7.42-7.19 (m, 5H), 4.0 (s, 2H),3.85 (s, 3H), 3.79 (s, 2H), 3.03 (bs, 2H), 2.91 (bs, 2H), 1.76 (bs, 4H).

Compound 723 was prepared from compound 722 using a procedure analogousto that for the preparation of compound 718 above. ¹H NMR (300 MHz,DMSO-d₆) δ 12.66 (s, 1H), 11.17 (s, 1H), 10.06 (s, 1H), 8.52-8.50 (m,1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.81-7.76 (m, 1H), 7.58-7.55 (d, 1H),7.42-7.19 (m, 4H), 6.99-6.96 (d, 1H), 4.0 (s, 2H), 3.70 (s, 2H), 3.03(bs, 2H), 2.91 (bs, 2H), 1.76 (bs, 4H).

Compound 1129 was prepared from 3-bromo-5-trifluoromethoxyanisole usinga procedure analogous to that for compound 1126 below.

Compound 729 was prepared using compound 1129 with a procedure analogousto that for compound 670. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66 (s, 1H),11.28 (s, 1H), 8.52-8.50 (m, 1H), 8.21-8.18 (d, J=9.06 Hz, 1H),7.81-7.76 (m, 1H), 7.58-7.55 (d, 1H), 7.42-7.29 (m, 2H), 6.99-6.95 (m,2H), 6.84 (s, 1H), 4.0 (s, 2H), 3.80 (m, 5H), 3.03 (bs, 2H), 2.91 (bs,2H), 1.76 (bs, 4H).

Compound 730 was prepared from compound 729 using a procedure analogousto that for the preparation of compound 718 above. ¹H NMR (300 MHz,DMSO-d₆) δ 12.66 (s, 1H), 11.28 (s, 1H), 10.04 (s, 1H), 8.52-8.50 (m,1H), 8.21-8.18 (d, J=9.06 Hz, 1H), 7.81-7.76 (m, 1H), 7.58-7.55 (d, 1H),7.42-7.29 (m, 2H), 6.81-6.78 (m, 2H), 6.61 (s, 1H), 4.0 (s, 2H), 3.74(m, 2H), 3.03 (bs, 2H), 2.91 (bs, 2H), 1.76 (bs, 4H).

To a mixture of 6-(di-Boc-amino)-2-bromopyridine (1 g, 2.9 mmol),bis(tri-tert-butylphosphine) palladium(0) (300 mg, 0.59 mmol) in1,4-dioxane (30 ml) under argon atmosphere was added 0.5 M of2-tert-butoxy-2-oxoethyl zinc chloride in ether (15 ml). The resultingmixture was stirred at room temperature overnight. The mixture waspartitioned between saturated NH₄Cl and EtOAc. The organic extract waswashed with brine, dried over sodium sulfate, filtered and evaporated.The crude material was purified by silica gel chromatography elutingwith 0-20% EtOAc in Hexane to afford 1123.

To a solution of 1123 (150 mg, 0.37 mmol) in MeOH (6 ml) and water (2ml) at 0° C. was added Lithium hydroxide monohydrate (100 mg, 2.38mmol). The resulting mixture was stirred at room temperature for 2 daysbefore it was evaporated to dryness. The mixture was then acidified with1N HCl (pH 4), and it was partitioned between water and EtOAc. Theorganic extract was washed with water, dried over sodium sulfate,filtered and evaporated to afford 1124.

A flask was charged with 657 (105 mg, 0.232 mmol), 1124 (90 mg, 0.255mmol) in DMF (1 ml) at 0° C. was added propylphosphonic anhydridesolution (300 ul) followed by triethylamine (89 ul, 0.64 mmol). Theresulting mixture was slowly warmed up to room temperature and stirredfor 3 h before it was quenched by addition of ice water (˜5 mL). Theprecipitate was collected by suction filtration, rinsed with more water.The crude material was purified by silica gel chromatography elutingwith 0-6% MeOH in DCM to afford 724. ¹H NMR (300 MHz, DMSO-d₆) δ 12.67(s, 1H), 11.32 (s, 1H), 9.69 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H),7.72-7.01 (m, 8H), 3.91-3.87 (d, 4H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.75(bs, 4H) 1.47 (s, 9H).

To a solution of 724 (50 mg, 0.07 mmol) in DCM (3 ml) at 0° C. was addedTFA (3 ml) dropwise. The resulting mixture was stirred at roomtemperature for 3 h before it was evaporated to dryness then trituratedthe residue with ether to afford 725. ¹H NMR (300 MHz, DMSO-d₆) δ 12.67(s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.88-7.77 (m, 3H),7.59-7.26 (m, 5H), 6.90-6.80 (m, 2H), 4.05 (s, 2H), 3.87 (s, 2H), 3.01(bs, 2H), 2.90 (bs, 2H), 1.75 (bs, 4H).

To a stirred solution of tert-butyl acetate (789 ul, 5.88 mmol),2-chloro-6-methylpyridine (428 ul, 3.92 mmol),chloro(2-di-t-butylphosphino-2′,4′,6′-tri-1-propyl-1,1′-bi-phenyl)[2-(2-aminoethyl)phenyl]palladium(II)(27 mg, 0.039 mmol) in toluene (10 ml) at 0° C. under argon was added asolution of LHMDS (1M in toluene) (12 ml, 12 mmol) pre-cooled to 0° C.The resulting mixture was stirred for 1 h. The mixture was partitionedbetween saturated NH₄Cl and EtOAc. The organic extract was washed withbrine, dried over sodium sulfate, filtered and evaporated. The crudematerial was purified by silica gel chromatography eluting with 0-15%EtOAc in Hexane to afford 1125.

To a solution of 1125 (267 mg, 1.29 mmol) in DCM (3 ml) at 0° C. wasadded TFA (1.5 ml) dropwise. The resulting mixture was stirred at roomtemperature overnight before it was evaporated to dryness thentriturated the residue with ether to afford 1126.

A flask was charged with 657 (50 mg, 0.111 mmol), 1126 (35 mg, 0.133mmol) in DMF (1 ml) at 0° C. was added propylphosphonic anhydridesolution (155 ul) followed by triethylamine (57 ul, 0.4 mmol). Theresulting mixture was slowly warmed up to room temperature and stirredfor 3 h before it was quenched by addition of ice water (˜5 mL). Theprecipitate was collected by suction filtration, rinsed with more water.The crude material was purified by silica gel chromatography elutingwith 0-6% MeOH in DCM to afford 728. ¹H NMR (300 MHz, DMSO-d₆) δ 12.67(s, 1H), 11.32 (s, 1H), 8.22-8.19 (d, J=9.12 Hz, 1H), 7.69-7.15 (m, 8H),3.96 (s, 2H), 3.87 (s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 2.52 (s, 3H),1.75 (bs, 4H).

To a solution of ethyl 2-pyridyl acetate (1 g, 6.05 mmol) in DCM (20 ml)at 0° C. was added MCPBA (77% max) (1.77 g, 10.2 mmol). The resultingmixture was warmed up to room temperature for 3 h before it waspartitioned between saturated sodium bicarbonate and DCM. The organicextract was washed with brine, dried over sodium sulfate, filtered andevaporated. The crude material was purified by silica gel chromatographyeluting with 0-12% MeOH in EtOAc to afford 1127.

To a suspension of 657 (331 mg, 0.73 mmol) in toluene was added 1127(278 mg, 1.53 mmol) followed by trimethylaluminum (2M in toluene) (732ul, 1.46 mmol). The resulting mixture was stirred at 60° C. overnight.The reaction mixture was partitioned between water and DCM. The organicextract was washed with brine, dried over sodium sulfate, filtered andevaporated. The crude material was purified by silica gel chromatographyeluting with 0-5% MeOH in DCM then 0-15% MeOH in EtOAc to afford 716. ¹HNMR (300 MHz, DMSO-d₆) δ 12.67 (s, 1H), 11.32 (s, 1H), 8.29-8.27 (m,1H), 8.21-8.19 (d, J=9.12 Hz, 1H), 7.61-7.26 (m, 8H), 4.03 (s, 2H), 3.87(s, 2H), 3.01 (bs, 2H), 2.90 (bs, 2H), 1.75 (bs, 4H).

Example 2: Compound Assays

Compounds were assayed in both an in vitro biochemical assay and a cellproliferation assay as follows. The IC50 results are provided in Table2.

Recombinant Enzyme Assay

Compounds were assessed for their ability to inhibit the enzymaticactivity of a recombinant form of Glutaminase 1 (GAC) using abiochemical assay that couples the production of glutamate (liberated byGAC) to glutamate dehydrogenase (GDH) and measuring the change inabsorbance for the reduction of NAD⁺ to NADH. Substrate solution wasprepared (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K₂HPO₄, 0.1 mg/mlBSA, 1 mM DTT, 20 mM L-glutamine, 2 mM NAD⁺, and 10 ppm antifoam) and 50μL added to a 96-well half area clear plate (Corning #3695). Compound (2μL) was added to give a final DMSO concentration of 2% at 2× the desiredconcentration of compound. Enzymatic reaction was started with theaddition of 50 μL of enzyme solution (50 mM Tris-HCl pH 8.0, 0.2 mMEDTA, 150 mM K₂HPO₄, 0.1 mg/ml BSA, 1 mM DTT, 10 ppm antifoam, 4units/ml GDH, 4 mM adenosine diphosphate, and 4 nM GAC) and read in aMolecular Devices M5 plate reader at 20° C. The plate reader wasconfigured to read absorbance (λ=340 nm) in kinetic mode for 15 minutes.Data was recorded as milli-absorbance units per minute and slopes werecompared to a control compound and a DMSO-only control on the sameplate. Compounds with slopes less than the DMSO control were consideredinhibitors and plate variability was assessed using the controlcompound.

Results from this assay for several compounds of the invention are shownin Table 2, expressed as IC50, or half maximal inhibitory concentration,wherein IC50 is a quantitative measure indicating how much compound isneeded to inhibit a given biological activity by half.

Recombinant Enzyme Assay—Time Dependence

Compounds were assessed for their ability to inhibit the enzymaticactivity of a recombinant form of Glutaminase 1 (GAC) using abiochemical assay that couples the production of glutamate (liberated byGAC) to glutamate dehydrogenase (GDH) and measuring the change inabsorbance for the reduction of NAD⁺ to NADH. Enzyme solution wasprepared (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K₂HPO₄, 0.1 mg/mlBSA, 1 mM DTT, 10 ppm antifoam, 4 units/ml GDH, 4 mM adenosinediphosphate, and 4 nM GAC) and 50 μL added to a 96-well half area clearplate (Corning #3695). Compound (2 μL) was added to give a final DMSOconcentration of 2% at 2× the desired concentration of compound. Theenzyme/compound mix was sealed with sealing foil (USA Scientific) andallowed to incubate, with mild agitation, for 60 minutes at 20° C.Enzymatic reaction was started with the addition of 50 μL of substratesolution (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K₂HPO₄, 0.1 mg/mlBSA, 1 mM DTT, 20 mM L-glutamine, 2 mM NAD⁺, and 10 ppm antifoam) andread in a Molecular Devices M5 plate reader at 20° C. The plate readerwas configured to read absorbance (λ=340 nm) in kinetic mode for 15minutes. Data was recorded as milli-absorbance units per minute andslopes were compared to a control compound and a DMSO-only control onthe same plate. Compounds with slopes less than the DMSO control wereconsidered inhibitors and plate variability was assessed using thecontrol compound.

Results from this assay for several compounds are shown in Table 2,expressed as IC50, or half maximal inhibitory concentration, whereinIC50 is a quantitative measure indicating how much compound is needed toinhibit a given biological activity by half.

Cell Proliferation Assay

P493-6 (myc “on”) cells were maintained in growth media (RPMI-1640, 10%FBS, 2 mM glutamine, 100 units/ml Penicillin and 100 μg/ml streptomycin)at 37° C. with 5% CO₂. For compound assay, P493-6 cells were plated in96-well V-bottom plates on the day of compound addition in 50 μl ofgrowth media at a cell density of 200,000 cells/ml (10,000 cells/well).Compounds were serially diluted in 100% DMSO at 200-times the finalconcentration. Compounds were diluted 100-fold into growth media andthen 50 μl of this mixture was added to cell plates making the finalconcentration of DMSO 0.5%. Cells were incubated with compound for 72hrs at 37° C. with 5% CO₂ and analyzed for antiproliferative effectseither by Cell Titer Glo (Promega) or FACS analysis using the Viacount(Millipore) kit on the Guava instrument.

Results from this assay for several compounds are shown in Table 2,expressed as IC50, or half maximal inhibitory concentration, whereinIC50 is a quantitative measure indicating how much compound is needed toinhibit a given biological activity by half.

Modified Recombinant Enzyme Assay—Time Dependence

Compounds were assessed for their ability to inhibit the enzymaticactivity of a recombinant form of glutaminase using a biochemical assaythat couples the production of Glu (liberated by glutaminase) to GDH andmeasures the increase in fluorescence due to the reduction of NADP+ toNADPH.

Assay Set-up: Glutaminase reaction buffer was prepared [50 mM Tris-HClpH 8.8, 150 mM K2HPO4, 0.25 mM EDTA, 0.1 mg/ml BSA (Calbiochem no.2960), 1 mM DTT, 2 mM NADP+(Sigma Aldrich no. N5755), and 0.01% TX-100]and used to make 3×-enzyme-containing solution, 3×-substrate-containingsolution, and 3×-inhibitor-containing solution (see below)Inhibitor-containing solution was made by diluting DMSO stocks ofcompounds into the glutaminase reaction buffer to create a 3× inhibitorsolution containing 6% DMSO. 3×-enzyme-containing solution was made bydiluting recombinant glutaminase and GDH from Proteus species (SigmaAldrich no. G4387) into glutaminase buffer to create a 6 nM glutaminaseplus 18 units/mL GDH solution. A 3× substrate solution containing eitherGln, Glu, or NADPH was made by diluting a stock of Gln (Sigma Aldrichno. 49419), Glu (Sigma Aldrich no. 49449), or NADPH (Sigma Aldrich no.N1630) into glutaminase reaction buffer to create a 3×-substratesolution. Reactions were assembled in a 384-well low-volume blackmicrotiter plates (Molecular Devices no. 0200-5202) by mixing 5 μL ofinhibitor-containing solution with 5 μL of substrate-containing solutionfollowed by 5 μL of enzyme-containing solution when no preincubation wasrequired. When time-dependent effects of compound inhibition weretested, enzyme-containing solution was treated with inhibitor-containingsolution for the indicated time prior to addition ofsubstrate-containing solution.

Measurement of glutaminase activity: Following the mixture of all threecomponents, fluorescence increase (Ex: 340 nM, Em:460 nm) was recordedfor 15 min at room temperature using the Spectromax M5e (MolecularDevices). IC50 Determination: The initial velocities of each progresscurve were calculated using a straight line equation(Y=Yintercept+(slope)*X). Initial velocity values were plotted againstcompound concentration and fit to a four parameter dose responseequation (% activity=Bottom+(Top−Bottom)/(1+10{circumflex over( )}((LogIC50−X)*HillSlope))) to calculate an IC50 value.

Results from this assay for several compounds are shown in Table 2,expressed as IC50, or half maximal inhibitory concentration, whereinIC50 is a quantitative measure indicating how much compound is needed toinhibit a given biological activity by half.

TABLE 2 Mod- ified GAC GAC Delta Delta GAC N2 N2 Delta Cell IC50 IC50 N2prolif 60 60 IC50 P493 min min no 72 h Cmpd preinc preinc preinc IC50 IDStructure (μM) (μM) (μM) (μM) 1

0.10 0.20 0.47 295

0.01 0.057 0.039 318

0.006 0.18 0.017 339

0.005 0.16 0.009 354

0.10 0.047 402

1.1 0.054 436

0.006 0.010 533

0.007 0.041 616

0.008 0.13 447

0.005 0.016 585

0.006 0.070 586

0.013 0.031 600

0.005 0.008 614

0.008 0.082 615

0.009 0.12 629

>20 0.065 636

0.008 0.059 657

0.24 1.5 658

0.005 0.040 659

0.010 0.058 660

0.025 0.037 661

0.007 0.12 662

0.007 0.055 663

0.007 0.089 666

0.004 0.058 668

0.009 0.026 669

0.021 0.026 670

0.005 0.030 671

0.004 0.035 672

0.010 0.045 673

0.006 0.033 674

0.008 0.024 675

0.040 676

0.030 677

0.056 678

0.026 679

0.036 680

0.033 681

0.019 682

0.017 683

0.024 684

0.042 685

0.022 686

0.010 687

0.011 688

0.012 689

0.013 690

0.017 692

0.020 693

0.070 694

0.029 695

0.030 696

0.034 697

0.050 698

0.098 699

0.12 700

0.17 701

0.11 702

0.31 703

0.012 704

0.88 705

0.032 706

14 707

0.085 708

2.8 709

0.14 710

711

712

713

714

715

0.19 0.39 716

0.18 717

0.034 0.19 718

0.026 0.015 719

0.033 0.01 720

0.020 0.92 721

0.016 0.022 722

0.024 0.016 723

0.042 0.02 724

0.14 0.034 725

0.050 0.15 726

0.54 0.61 727

0.023 0.012 728

0.012 0.018 729

0.016 0.026 730

0.013 0.025

Example 3: Caco-2 Permeability Assay

Caco-2 cells are commonly used in a confluent monolayer on a cellculture insert filter. When cultured in this format and under specificconditions, the cells become differentiated and polarized such thattheir phenotype, morphologically and functionally resembles theenterocytes lining the small intestine. The cell monolayer provides aphysical and biochemical barrier to the passage of small molecules, andis widely used across the pharmaceutical industry as an in vitro modelof the human small intestinal mucosa to predict the absorption of orallyadministered drugs (Hidalgo et al., Gastroenterology, 1989; Artursson,J. Pharm. Sci., 1990). The correlation between the in vitro apparentpermeability (P

app) across Caco-2 monolayers and the in vivo absorption is wellestablished (Artursson et al., Biochem. Biophys. Res. Comm., 1991).

The present assay was used to determine the bidirectional permeabilityof the compounds of the invention through Caco-2 cell monolayers. Caco-2cells were grown in confluent monolayers where the media of both theapical (A) and basolateral (B) sides were at pH 7.4. Compounds weredosed at 1 μM in the presence of 200 μM Lucifer Yellow, on the apicalside (A→B) or the basolateral side (B→A) for assessment, in duplicate.Samples from both A and B sides were taken after 120 minutes exposure,and compound concentration (reported as percent recovery) was determinedusing a generic LC-MS/MS method with a minimum four-point calibrationcurve.

The absorption potential of compounds were classified as either Low(P-app<1×10⁻⁶ cm/s) or High (P-app>1×10⁻⁶ cm/s). The efflux ratio wascalculated as (Papp B→A)/(Papp A→B), with efflux ratios beingsignificant when greater than or equal to 3 when the Papp (B→A) wasgreater than or equal to 1×10⁻⁶ cm/s. Results for certain compounds ofthe invention are shown in Table 3.

TABLE 3 Caco-2 Permeability Results Recovery Papp Efflux PermeabilitySignificant Cmpd Direction (%) (avg.) Ratio Classification Efflux 533A→B 41 4.94 7.6 High Yes B→A 52 37.5 585 A→B 42 7.52 3.1 High Yes B→A 5323.4 616 A→B 65 8.23 6.0 High Yes B→A 76 49.5 295 A→B 89 8.17 7.3 HighYes B→A 96 59.8 318 A→B 73 2.45 18 High Yes B→A 82 44.5 339 A→B 73 2.3917 High Yes B→A 80 41.6 354 A→B 117 1.38 33 High Yes B→A 101 45.0 436A→B 44 3.75 6.6 High Yes B→A 57 24.7 660 A→B 56 0.61 3.9 Low Yes B→A 682.37 670 A→B 70 9.64 6.2 High Yes B→A 72 59.6 679 A→B 34 7.59 2.6 HighNo B→A 42 19.6 447 A→B 71 7.76 3.5 High Yes B→A 56 27.2 703 A→B 51 6.266.6 High Yes B→A 66 41.0 705 A→B 60 8.52 6.0 High Yes B→A 67 51.0

Example 4: Solubility

Ca. 1 mg portions of test article were combined with 120 μL solvent inwells of a 96×2 mL polypropylene plate. The plate was vigorously vortexmixed at room temperature (ca. 20 C) for 18 hr and each well checkedvisually for undissolved solid; wells containing no visible solid werecharged with additional solid test article and vortex mixed another 6 hrat room temperature after which all wells showed visible solid. Thecontents of all wells were then filtered through a 0.45 μm GHP filterplate to yield clear filtrates. 5 μL of each filtrate was diluted into100 μL DMF and vortex mixed to yield HPLC samples. Duplicatequantitation standards for each test article were prepared by dilutingweighed portions of solid test article in measured volumes of DMF. 2 μLof each HPLC sample and quantitation standard were analyzed by HPLCusing the method outlined in Table 4. Dissolved test articleconcentrations were calculated by peak area ratio against theappropriate quantitation standards. Solubility results are presented inTable 5.

TABLE 4 Outline of HPLC Method Instrument Shimadzu Prominence UFLC withDiode Array UV/Vis Detector Column VWR Sonoma C8(2), 3.5 μm, 2.1 × 50 mmColumn 40° C. Temp Mobile 0.1% (v/v) formic acid in water Phase A Mobile0.1% (v/v) formic acid in acetonitrile Phase B Flow Rate 0.4 mL/min TimeGradient (min) % Mobile Phase B 0 20 8 100 8.5 100 8.6 20 9.6 END

TABLE 5 Measured Solubilities Solubility (mg/mL) Solvent 1 295 402 585670 447 703 water <0.002 <0.002 <0.004 <0.002 0.007 <0.004 <0.004 0.9%NaCl <0.002 <0.002 <0.004 <0.002 <0.002 0.005 <0.004 0.1M HCl <0.0020.003 <0.004 <0.002 0.005 <0.004 <0.004 50 mM Cit <0.002 <0.002 <0.004<0.002 0.066 <0.004 <0.004 pH 2.3 50 mM Cit <0.002 <0.002 <0.004 <0.0020.003 <0.004 <0.004 pH 3.3 50 mM Cit <0.002 <0.002 <0.004 <0.002 <0.002<0.004 <0.004 pH 4.4 50 mM Cit <0.002 <0.002 <0.004 <0.002 <0.002 <0.004<0.004 pH 5.4 PBS <0.002 <0.002 <0.004 <0.002 <0.002 <0.004 <0.004 0.1M14.420 0.268 <0.004 0.192 0.227 0.192 0.656 NaOH 10% PS80/ 0.050 0.0270.153 0.261 1.204 0.851 0.378 50 mM cit 10% CrEL/ 0.076 0.055 0.1570.228 0.458 0.732 0.309 50 mM cit 20% 0.046 0.090 0.019 0.125 5.2562.718 0.476 SBECD/ 50 mM cit 20% 0.042 0.167 0.056 0.327 9.685 2.1770.651 HPBCD/ 50 mM cit Labrasol 0.258 0.918 31.032 5.004 5.042 77.16420.727 Capryol 0.042 1.540 11.210 1.780 1.519 7.916 3.683 PGMC Capryol90 0.081 0.215 13.676 1.744 1.974 11.114 7.409 canola oil <0.002 <0.0020.529 0.072 0.012 0.071 0.014 PEG400 0.451 1.644 30.179 3.944 9.90157.334 22.419 PG 0.048 0.234 1.365 1.422 2.569 8.265 4.698 EtOH 0.0400.083 2.958 1.991 0.964 3.921 2.645

Example 5: Pharmacokinetic Studies

For PK studies performed in mice, female CD-1 mice, age 5-8 weeks, weredosed orally by gavage (10 mL/kg volume) with test compounds. Atpre-determined time-points (e.g. 1, 3, and 6 hrs) post-dosing, groups ofthree mice were sacrificed by CO₂ inhalation and blood collected viacardiac puncture. Blood samples were placed into K2 EDTA coated tubesand kept on ice. Samples were centrifuged at 2000×g for 10 minutes at 4°C. and plasma isolated. Plasma was stored at ˜70° C. until bioanalyticalanalysis. FIGS. 1 and 2 show the plasma concentration of compounds 585,295, 447 and 318 over time following oral dosing of 50 mg/kg to femaleCD-1 mice.

For PK studies performed in rats, groups of three female Sprague-DawleyRats, age 7-10 weeks, with jugular vein cannulae were dosed orally bygavage (5-10 mL/kg) with test compounds. Serial blood samples werecollected via the jugular vein cannulae at multiple time-points pre andpost-dose. Blood samples were placed into K2 EDTA coated tubes on kepton ice. Samples were centrifuged at 2500×g for 10 minutes at 4° C. andplasma isolated. Plasma was stored at ˜70° C. until bioanalyticalanalysis. FIG. 3 shows the plasma concentration of compound 670 overtime following oral dosing of 500, 250, 80 and 25 mg/kg in 25%Hydroxypropyl-β-cyclodextrin (HPBCD formulation), to female SpragueDawley rats.

Results from several studies are shown in Table 6. The plasma sampleswere analyzed using Liquid chromatography tandem mass spectrometry(LC/MS/MS) methods. Frozen plasma samples were thawed on ice or roomtemperature. Aliquotes of study plasma samples and calibration samplesprepared in the same matrix as the study samples were quenched in anorganic solvent (methanol, acetonitrole or DMF) containing an internalstandard. The mixtures were then centrifuged and filtered and thefiltrates were injected to an LC/MS/MS system to determineconcentrations of test compounds.

TABLE 6 Pharmacokinetics studies and results Solution/ Time- DoseSuspen- points Cmax Tmax Cmpd (mg/kg) Species Vehicle sion (h) (uM) (h)295 50 Mouse Eudragit Suspen- 1, 6 0.02  6 L100-55 sion solid dispersion585 50 Mouse Eudragit Suspen- 0.5, 1, 0.93  3 L100-55 sion 3, 6 soliddispersion 585 50 Mouse 1% NMP/ Solution 0.5, 1, 5.16  1 99% 3, 6Gelucire 585 50 Rat Eudragit Suspen- 0.5, 1, 1.15  2 L100-55 sion 2, 4,solid 8, 24 dispersion 670 25 Rat 20% Solution pre, 0.5, 4.4   1 HPBCD/1, 2, 10 mM 4, 8, Citrate 24 pH 2.0 670 80 Rat 65% Solution pre, 0.5,12.5   1 HPBCD/ 1, 2, 10 mM 4, 8, Citrate 24 pH 2.6 670 250 Rat 40%Solution pre, 0.5, 9.9  24 HPBCD/ 1, 2, 10 mM 4, 8, Citrate 24 pH 2.2670 500 Rat 40% Solution pre, 0.5, 15.3   2 HPBCD/ 1, 2, 10 mM 4, 8,Citrate 24 pH 2.2 670 100 Mouse 25% Solution 1, 2, 4, 5.75  1 HPBCD/ 8,24 10 mM Citrate pH 2.2 670 200 Mouse 25% Solution 1, 2, 4, 5.05  1HPBCD/ 8, 24 10 mM Citrate pH 2.2 447 50 Mouse 2% NMP/ Solution 1, 3, 61.13  1 98% Gelucire 318 50 Mouse 0.5% Suspen- 0.5, 1, 0.01  1 CMC/ sion3, 6 0.1% PS80 318 50 Mouse Capryol Suspen- 0.5, 1, 0.08  3 PGMC sion 3,6

Example 6: Lung Adenocarcinoma Xenograft Efficacy Study

Female scid/beige mice (n=20) age 6-8 weeks were implantedsubcutaneously with 1×10⁷ H2122 lung adenocarcinoma cells per mousesuspended in PBS. Mice were randomized into the following two groups ofn=10 mice/group: 1) Vehicle control (25% Hydroxypropyl-β-cyclodextrin)and 2) Compound 670 dosed orally at 200 mg/kg (formulated at 20 mg/mL in25% HP-β-CD). For both groups, dosing was initiated 24 hourspost-implant and continued orally BID for 23 days. Tumors were measuredwith calipers three times per week and tumor volume calculated using theformula tumor volume (mm³)=(a×b²/2) where ‘b’ is the smallest diameterand ‘a’ is the largest perpendicular diameter. **P-value <0.01(Two-sided T-test). Results are shown in FIG. 4.

Example 7: Triple Negative Breast Cancer Xenograft Study

Female CB.17 SCID mice (n=40) age 8-12 weeks were implantedsubcutaneously with 1×10⁷ JIMT-1 triple negative breast cancer cells permouse mixed 1:1 with Matrigel. When tumor volumes reached 100-150 mm³mice were randomized into the following four groups of n=10mice/group: 1) Vehicle control (25% Hydroxypropyl-β-cyclodextrin) dosedorally BID for 35 days; 2) Compound 670 dosed orally at 200 mg/kg BIDfor 35 days (formulated at 20 mg/mL in 25% HP-β-CD); 3) Paclitaxel dosedintravenously at 10 mg/kg every other day for a total of 5 doses; and 4)Compound 670 (200 mg/kg orally BID×35 days) and Paclitaxel (10 mg/kg IVgod×5). Tumors were measured with calipers two times per week and tumorvolume calculated using the formula tumor volume (mm³)=(a×b²/2) where‘b’ is the smallest diameter and ‘a’ is the largest perpendiculardiameter. **P-value <0.01 (One-way ANOVA vs. vehicle). ## P-value <0.01(Two-sided T-test vs. paclitaxel alone). Results are shown in FIG. 5.

Example 8: Multiple Myeloma Xenograft Study

Female CB.17 SCID mice (n=20) age 8-12 weeks were implantedsubcutaneously with 1×10⁷ RPMI-8226 myeloma cells per mouse mixed 1:1with Matrigel. Mice were randomized into the following two groups ofn=10 mice/group: 1) Vehicle control (25% Hydroxypropyl-β-cyclodextrin)and 2) Compound 670 dosed at orally at 200 mg/kg (formulated at 20 mg/mLin 25% HP-β-CD). For both groups, dosing was initiated when tumorsreached a volume of 100-150 mm³ and continued orally BID for 28 days.Tumors were measured with calipers two times per week and tumor volumecalculated using the formula tumor volume (mm³)=(a×b²/2) where ‘b’ isthe smallest diameter and ‘a’ is the largest perpendicular diameter.**P-value <0.01 (Two-sided T-test). Results are shown in FIG. 6.

Example 9: Treatment of Multiple Myeloma Cells with a Combination ofDrugs

As shown in FIG. 7, MM1S cells (panels A & B) and RPMI-8226 cells(panels C & D) were treated with a dose titration of either compound670, pomalidomide or a mixture thereof (panels A & C) or compound 670,dexamethsone or a mixture thereof (panels B & D) for 72 hours in growthmedia. At the end of the incubation, cell viability was measured usingCell Titer Glo as per manufacturer's protocol (Promega, Madison, Wis.).Measured values for compound-treated cells were normalized toDMSO-treated cells and data is reported as a cell survival ratio with avalue of 1 (one) corresponding to maximum cell survival and a value of 0(zero) corresponding to no cell survival. Cell survival ratios for allcompound treatments are represented as bar graphs. Combination indiceswere calculated using the Calcusyn program (biosoft.com) and reportedfor individual mixtures of compound 670 and pomalidomide [POM] (panels A&C) and individual mixtures of compound 670 and dexamethasone [DEX](panels B & D). Compound mixtures that produced a synergistic anti-tumoractivity are highlighted.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

The invention claimed is:
 1. A compound of formula I,

or a pharmaceutically acceptable salt thereof, wherein: L representsCH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂, CH═CH, or

 wherein any hydrogen atom of a CH or CH₂ unit may be replaced by alkylor alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and anyhydrogen atom of a CH₂ unit of CH₂CH₂, CH₂CH₂CH₂ or CH₂ may be replacedby hydroxy; X, independently for each occurrence, represents S, O orCH═CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;Y, independently for each occurrence, represents H or CH₂O(CO)R₇; R₇,independently for each occurrence, represents H or substituted orunsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy; Z represents H orR₃(CO); R₁ and R₂ each independently represent H, alkyl, alkoxy orhydroxy; R₃ represents substituted or unsubstituted alkyl, hydroxyalkyl,aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀), N(R₄)(R₅) or OR₆,wherein any free hydroxyl group may be acylated to form C(O)R₇, R₄ andR₅ each independently for each occurrence represent H or substituted orunsubstituted alkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl,alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,wherein any free hydroxyl group may be acylated to form C(O)R₇; R₆represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl,acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy,aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, orheteroaryloxyalkyl, wherein any free hydroxyl group may be acylated toform C(O)R₇; R₈, R₉ and R₁₀ each independently for each occurrencerepresent H or substituted or unsubstituted alkyl, hydroxy,hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl,alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, or heteroaryloxyalkyl, or R₈ and R₉ together with thecarbon to which they are attached, form a carbocyclic or heterocyclicring system, wherein any free hydroxyl group may be acylated to formC(O)R₇, and wherein at least two of R₈, R₉ and R₁₀ are not H; R₁₁represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the arylor heteroaryl ring is substituted with either —OCHF₂ or —OCF₃ and isoptionally further substituted, or R₁₁ represents C(R₁₂)(R₁₃)(R₁₄),N(R₄)(R₁₄) or OR₁₄, wherein any free hydroxyl group may be acylated toform C(O)R₇; R₁₂ and R₁₃ each independently represent H or substitutedor unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,wherein any free hydroxyl group may be acylated to form C(O)R₇, andwherein both of R₁₂ and R₁₃ are not H; and R₁₄ represents aryl,arylalkyl, aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, or heteroaryloxyalkyl, wherein the aryl or heteroarylring is substituted with either —OCHF₂ or —OCF₃ and is optionallyfurther substituted.
 2. The compound of claim 1, wherein the compound isnot one of the following:


3. The compound of claim 1, wherein R₁₁ represents arylalkyl, whereinthe aryl ring is substituted with —OCF₃.
 4. The compound of claim 3,wherein R₁₁ represents trifluoromethoxybenzyl.
 5. The compound of claim4, wherein R₁₁ represents


6. The compound of claim 1, wherein L represents CH₂SCH₂, CH₂CH₂, CH₂Sor SCH₂.
 7. The compound of claim 6, wherein L represents CH₂CH₂.
 8. Thecompound of claim 1, wherein each Y represents H.
 9. The compound ofclaim 1, wherein X represents S or CH═CH, wherein any hydrogen atom of aCH unit may be replaced by alkyl.
 10. The compound of any one of claims1-9, wherein Z represents R₃(CO).
 11. The compound of claim 10, whereinR₃ and R₁₁ are not identical.
 12. The compound of claim 1, wherein R₁and R₂ each represent H.
 13. The compound of claim 10, wherein Zrepresents R₃(CO) and R₃ represents substituted or unsubstitutedarylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
 14. Thecompound of claim 13, wherein Z represents R₃(CO) and R₃ representssubstituted or unsubstituted heteroarylalkyl.
 15. The compound of claim14, wherein Z represents R₃(CO) and R₃ represents substituted orunsubstituted pyridylalkyl.
 16. A compound having the structure

or a pharmaceutically acceptable salt thereof.
 17. A pharmaceuticalcomposition comprising one or more pharmaceutically acceptableexcipients and a compound of formula I,

or a pharmaceutically acceptable salt thereof, wherein: L representsCH₂SCH₂, CH₂CH₂, CH₂CH₂CH₂, CH₂, CH₂S, SCH₂, CH₂NHCH₂, CH═CH, or

 wherein any hydrogen atom of a CH or CH₂ unit may be replaced by alkylor alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and anyhydrogen atom of a CH₂ unit of CH₂CH₂, CH₂CH₂CH₂ or CH₂ may be replacedby hydroxy; X, independently for each occurrence, represents S, O orCH═CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;Y, independently for each occurrence, represents H or CH₂O(CO)R₇; R₇,independently for each occurrence, represents H or substituted orunsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl,heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy; Z represents H orR₃(CO); R₁ and R₂ each independently represent H, alkyl, alkoxy orhydroxy; R₃ represents substituted or unsubstituted alkyl, hydroxyalkyl,aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, heteroaryloxyalkyl or C(R₈)(R₉)(R₁₀), N(R₄)(R₅) or OR₆,wherein any free hydroxyl group may be acylated to form C(O)R₇, R₄ andR₅ each independently for each occurrence represent H or substituted orunsubstituted alkyl, hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl,alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,wherein any free hydroxyl group may be acylated to form C(O)R₇; R₆represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl,acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy,aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, orheteroaryloxyalkyl, wherein any free hydroxyl group may be acylated toform C(O)R₇; R₈, R₉ and R₁₀ each independently for each occurrencerepresent H or substituted or unsubstituted alkyl, hydroxy,hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl,alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl,arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, or heteroaryloxyalkyl, or R₈ and R₉ together with thecarbon to which they are attached, form a carbocyclic or heterocyclicring system, wherein any free hydroxyl group may be acylated to formC(O)R₇, and wherein at least two of R₈, R₉ and R₁₀ are not H; R₁₁represents aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl,heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, wherein the arylor heteroaryl ring is substituted with either OCHF₂ or OCF₃ and isoptionally further substituted, or R₁₁ represents C(R₁₂)(R₁₃)(R₁₄),N(R₄)(R₁₄) or OR₁₄, wherein any free hydroxyl group may be acylated toform C(O)R₇, R₁₂ and R₁₃ each independently represent H or substitutedor unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino,aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino,alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl,cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl,wherein any free hydroxyl group may be acylated to form C(O)R₇, andwherein both of R₁₂ and R₁₃ are not H; and R₁₄ represents aryl,arylalkyl, aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl,heteroaryloxy, or heteroaryloxyalkyl, wherein the aryl or heteroarylring is substituted with either —OCHF₂ or —OCF₃ and is optionallyfurther substituted.
 18. The pharmaceutical composition of claim 17,wherein the compound is not one of the following:


19. The pharmaceutical composition of claim 17, wherein R₁₁ representsarylalkyl, wherein the aryl ring is substituted with —OCF₃.
 20. Thepharmaceutical composition of claim 19, wherein R₁₁ representstrifluoromethoxybenzyl.
 21. The pharmaceutical composition of claim 20,wherein R₁₁ represents


22. The pharmaceutical composition of claim 17, wherein L representsCH₂SCH₂, CH₂CH₂, CH₂S or SCH₂.
 23. The pharmaceutical composition ofclaim 22, wherein L represents CH₂CH₂.
 24. The pharmaceuticalcomposition of claim 17, wherein each Y represents H.
 25. Thepharmaceutical composition of claim 17, wherein X represents S or CH═CH,wherein any hydrogen atom of a CH unit may be replaced by alkyl.
 26. Thepharmaceutical composition of any one of claims 17-25, wherein Zrepresents R₃(CO).
 27. The pharmaceutical composition of claim 26,wherein R₃ and R₁₁ are not identical.
 28. The pharmaceutical compositionof claim 17, wherein R₁ and R₂ each represent H.
 29. The pharmaceuticalcomposition of claim 26, wherein Z represents R₃(CO) and R₃ representssubstituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl orheterocycloalkyl.
 30. The pharmaceutical composition of claim 29,wherein Z represents R₃(CO) and R₃ represents substituted orunsubstituted heteroarylalkyl.
 31. The pharmaceutical composition ofclaim 30, wherein Z represents R₃(CO) and R₃ represents substituted orunsubstituted pyridylalkyl.